GPCR-expressing cell lines

ABSTRACT

The present invention provides expression vectors that facilitate high levels of expression of GPCR proteins. Encompassed by the invention are methods and compositions for recombinant cell lines expressing GPCR proteins with the aid of the expression vectors of the instant invention. The recombinant cell lines of the instant invention express GPCR proteins at levels of at least about 150,000 copies of the protein per cell. The present invention also provides methods and compositions for raising antibodies against GPCR proteins using the high expressing recombinant cells of the instant invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of Provisional Patent ApplicationsNo. 60/730,997, filed Oct. 28, 2005, the disclosure of which isincorporated by reference herein in its entirety for all purposes.

BACKGROUND OF THE INVENTION

This invention relates generally to the field of G protein coupledreceptor (GPCR) expression and modulation.

G protein-coupled receptors (GPCRs) are a historically successfultherapeutic target family, with GPCR-directed drugs covering a widerange of therapeutic indications. As cell surface receptors, GPCRs arevital to cellular functioning, because they are primary mediators ofcell to cell communication.

Mammalian cells express very low levels of endogenous GPCRs, generallywith no more than three thousand copies per cell. This level issufficient for receptor function, but offers a challenge to GPCRresearch, which often requires much higher concentrations of functionalproteins. For example, structural studies, small molecule drug designand generation of functional antibodies against the native GPCRconformation require expression levels that are orders of magnitudehigher than what is seen using current methods.

Attempts have been made to isolate mammalian cell lines that overexpressexogenous GPCRs, but these past attempts have failed due to the cellulartoxicity that occurs with receptor overexpression. Attempts to createexpression systems in “lower” organisms have similarly met with limitedsuccess due to inefficient folding (bacteria), low yield (yeast) orincorrect post-translation modification (baculovirus).

A need thus exists for stable, high-expression systems capable ofproviding multiple copies of GPCR proteins for structural and functionalstudies.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides a novel method fordevelopment of mammalian cell lines that overexpress G protein coupledreceptor (GPCR) proteins. Exemplary cell lines of the invention expressGPCR at levels upwards of one million copies per cell. Such high levelsof expression are surprising, given that conventional methods ofexpression yield much lower levels of expression for transmembraneproteins.

In a first aspect, the present invention provides a vector forfacilitating high levels of expression of GPCR proteins in a cell line.The vector includes components such as a cytomegalovirus (CMV) promoter,a signal peptide, and epitope tag, a Kozak sequence, a poly-A site, anda viral origin of replication.

In second aspect, the invention provides a recombinant cell line whichexpresses GPCR proteins at a level of at least 150,000 copies per cell.In a further aspect, the invention provides methods for producingrecombinant cell lines by transfecting a host cell with at least oneexpression vector. In a preferred embodiment of the invention, theexpression vector may include a nucleotide sequence selected from SEQ IDNO: 19 and SEQ ID NO: 20.

In a still further aspect, the invention provides methods for usingrecombinant cell lines to screen for therapeutic candidates able tointeract with a GPCR protein. The method includes expressing a GPCRamino acid sequence in a recombinant cell. A test entity is contactedwith a region of the GPCR amino acid sequence, and this region presentsa fragment of the GPCR amino acid sequence that is sufficient for thetest entity to interact with the fragment. In an embodiment of theinvention, the test entity interacts with the fragment in a detectablemanner. Detection of the interaction between the test entity and thefragment of the GPCR amino acid sequence identifies the test entity as atherapeutic candidate.

In a still further aspect, the invention provides a method of using aGPCR-expressing cell line to identify a test compound which modulatesthe activity of the GPCR protein. This method includes making a firstmeasurement, which involves measuring second messenger activity in thecell line in the absence of the test compound, and making a secondmeasurement, which involves measuring second messenger activity in thepresence of the test compound. The method encompasses a comparison ofthe first and second measurement to determine if there is a differencebetween the two. A difference between the first and second measurementidentifies the test compound as a compound that modulates the activityof the GPCR. In a preferred embodiment of the invention, the cell lineexpresses at least 150,000 copies of the GPCR protein per cell.

In another aspect, the invention provides an antibody or antigen bindingfragment that is able to bind to a structural feature of a GPCR protein.In a further aspect of the invention, the antibody or antigen bindingfragment is raised against an immunogen which is a cell line expressingbetween 150,000 and 2,000,000 copies of GPCR protein per cell.

In a further aspect of the invention, a method is provided whereby cellsexpressing GPCR proteins are detected in a test sample. The test sampleis contacted with an antibody specifically binding to a structuralfeature of a GPCR protein. Specific binding of the antibody to astructural feature of a GPCR protein identifies the presence ofGPCR-expressing cells in the test sample. This method further includesthe detection of specific binding of the antibody to a structuralfeature of a GPCR protein.

In a still further aspect of the invention, a method is provided forproducing monoclonal antibodies for a GPCR protein. In this aspect ofthe invention, a test animal is immunized with at least one cell lineexpressing a GPCR protein, and the cell line preferably expresses atleast 50,000 copies of said GPCR protein per cell. The test animal isinduced to produce hybridomas, and the method includes isolating thehybridomas and screening for monoclonal antibodies using one or morecell-based assay systems.

In another aspect, the invention provides a kit for high throughputpurification and quantification of recombinant proteins of one or moremembers of one or more GPCR families. A kit according to the inventioncomprises a vector for expressing the recombinant proteins at levelsbetween 50,000 and 2,000,000 copies per cell. A kit according to theinvention can also comprise an affinity chromatography resin, aproteolytic enzyme, an internal quantification standard, a matrix forMALDI-TOF mass spectrometry, as well as instructions for use of the kit.

In still another aspect, the invention provides a method for identifyinga DNA sequence encoding a member of a GPCR family. This method includesthe process of probing a cDNA library or genomic library with a labeledprobe and identifying from the library sequences able to hybridize tothe probe under stringent conditions. Encompassed in the scope of theinvention are labeled probes comprising nucleotide sequences selectedfrom SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15 and 17.

In yet another aspect, the invention provides a method for producing afunctional assay cell line. This method includes producing a cell lineexpressing a GPCR protein and coupling a functional reporter to bindingof a ligand to the GPCR protein. The functional reporter is such that abinding event between the ligand and the GPCR protein is detectable as areporter activity readout. In an exemplary embodiment of the invention,the cell line expresses a GPCR protein comprising an amino acid sequenceselected from SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, and SEQ IDNO: 17. In a preferred embodiment of the invention, the cell lineexpresses at least 150,000 copies of the GPCR protein per cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 displays the amino acid sequence (SEQ ID NO: 1) and thenucleotide sequence (SEQ ID NO: 2) for the G-protein coupled receptorC5AR.

FIG. 2 displays the amino acid sequence (SEQ ID NO: 3) and thenucleotide sequence (SEQ ID NO: 4) for the G-protein coupled receptorNMUR1.

FIG. 3 displays the amino acid sequence (SEQ ID NO: 5) and thenucleotide sequence (SEQ ID NO: 6) for the G-protein coupled receptorP2RY2.

FIG. 4 displays the amino acid sequence (SEQ ID NO: 7) and thenucleotide sequence (SEQ ID NO: 8) for the G-protein coupled receptorPTAFR.

FIG. 5 displays the amino acid sequence (SEQ ID NO: 9) and thenucleotide sequence (SEQ ID NO: 10) for the G-protein coupled receptorAGTRL1.

FIG. 6 displays the amino acid sequence (SEQ ID NO: 11) and thenucleotide sequence (SEQ ID NO: 12) for the G-protein coupled receptorC3AR.

FIG. 7 displays the amino acid sequence (SEQ ID NO: 13) and thenucleotide sequence (SEQ ID NO: 14) for the G-protein coupled receptorCCR5.

FIG. 8 displays the amino acid sequence (SEQ ID NO: 15) and thenucleotide sequence (SEQ ID NO: 16) for the G-protein coupled receptorCXCR4.

FIG. 9 displays the amino acid sequence (SEQ ID NO: 17) and thenucleotide sequence (SEQ ID NO: 18) for the G-protein coupled receptorPAR2.

FIG. 10 is a schematic map of the features of the GPCR expression vectorpMEX2.

FIG. 11A and FIG. 11B show the nucleotide sequence of the GPCRexpression vector pMEX2.

FIG. 12 is a schematic map of the features of the GPCR expression vectorpMEX5.

FIG. 13A and FIG. 13B show the nucleotide sequence of the GPCRexpression vector pMEX5.

FIG. 14 displays the results of an ELISA assay of mouse immune seracollected after 3 immunizations with CXCR4 transfected cells as theimmunogen.

FIG. 15 displays data from FITC analysis of cell surface expression forthe identified G-protein coupled receptors (GPCRs).

FIG. 16 provides a surface expression profile of 72 recombinant GPCRs asdetermined by FACS analysis.

FIG. 17 provides data from a binding assay of transiently expressedhistamine receptors (H2).

FIG. 18 provides data from a cell surface assay for the identified GPCRstransfected into HEK293T cells.

FIG. 19 displays data from a calcium signaling assay for the GPCRs EDG4(CHO cells) and NMUR1 (HEK293T cells). CHO/Flag-EDG4 is a stable cellline used as an immunogen. The traces in the top row are from negativeantiserum tested by Flag peptide ELISA. The traces in the bottom row arefrom positive antiserum tested by Flag peptide ELISA. The traces labeled293/Flag-GPR40 are data from a stable cell line. 293/Flag-EDG4 and293/myc-EDG4 are data from transiently transfected cells.

FIG. 20 is data from FACS analysis of mouse immune sera where EDG4 CHOstable cell line was the immunogen. The dark black trace is from aCHO/GPR40 cell line, while the light gray trace is from a CHO/EDG4 cellline.

FIG. 21 displays a screening assay of anti-EDG4 monoclonal antibodiesfrom CHO/GPR40 and CHO/EDG4 cell lines.

FIG. 22 is a competitive binding assay using EDG4 antibody to block thereceptor activation by the G-protein 5G3.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The abbreviation “GPCR” refers to G-protein Coupled Receptor, and asused herein encompasses the protein, amino acid sequence, and nucleotidesequence encoding for a G-protein coupled receptor.

The terms “heterologous protein”, “recombinant protein”, and “exogenousprotein” can be used interchangeably in referring to aspects of thisinvention. These phrases refer to a polypeptide which is produced byrecombinant DNA techniques, wherein DNA encoding the polypeptide isinserted into a suitable expression vector which is in turn used totransform a host cell to produce the heterologous protein.

As used herein, “heterologous G protein coupled receptor” (e.g., aheterologous adenosine receptor) is a receptor encoded by heterologousDNA. Upon expression of the heterologous DNA in a recombinant cell, theheterologous receptor is expressed in the recombinant cell. The termheterologous G protein coupled receptor, or GPCR, as used hereinencompasses wildtype proteins (and the nucleotide sequences which encodefor them) as well as all variants or mutants, whether those variationsor mutations are naturally-occurring or created through genetic ormolecular engineering.

The term “signal transduction” encompasses the processing of physical orchemical signals from the extracellular environment through the cellmembrane and into the cell, and may occur through one or more of severalmechanisms, such as activation/inactivation of enzymes (such asproteases, or other enzymes which may alter phosphorylation patterns orother post-translational modifications), activation of ion channels orintracellular ion stores, effector enzyme activation via guaninenucleotide binding protein intermediates, formation of inositolphosphate, activation or inactivation of adenylyl cyclase, directactivation (or inhibition) of a transcriptional factor and/oractivation.

The term “functionally” couples to (as in a receptor that is“functionally integrated into a signaling pathway in a cell” or“functionally integrated into an endogenous yeast signaling pathway” or“functionally expressed by a host cell”) refers to the ability of areceptor to bind to modulators and transduce that binding event into asignal using components of a signaling pathway of the cell. For example,GPCR which is functionally integrated into an endogenous pheromoneresponse or signaling pathway of a yeast cell is expressed on thesurface of the yeast cell, couples to a G protein within the yeast celland transduces a signal in that yeast cell upon binding of a modulatorto the receptor.

The term “modulation”, as in “modulation of a (heterologous) G proteincoupled receptor” and “modulation of a signal transduction activity of areceptor protein” encompasses, in its various grammatical forms,induction and/or potentiation, as well as inhibition and/ordownregulation of receptor activity and/or one or more signaltransduction pathways downstream of a receptor.

An “oligonucleotide”, as used herein, refers to a stretch of nucleotideresidues which preferably has a sufficient number of bases to be used asan oligomer, amplimer or probe in a polymerase chain reaction (PCR).Oligonucleotides are prepared synthetically or from genomic or cDNAsequences and are preferably used to amplify, reveal, or confirm thepresence of a similar DNA or RNA in a particular cell or tissue.Oligonucleotides or oligomers comprise portions of a DNA sequence havingat least about 10 nucleotides and as many as about 35 nucleotides,preferably about 25 nucleotides.

“Probes” refers to oligonucleotides derived from naturally occurringrecombinant single- or double-stranded nucleic acids or may bechemically synthetic. Oligonucleotides are useful in detecting thepresence of complementary identical or similar sequences. Probes may belabeled with reporter molecules using nick translation, Klenow fill-inreaction, PCR or other methods well known in the art. Nucleic acidprobes may be used in Southern, Northern or in situ hybridization todetermine whether DNA or RNA encoding a certain protein is present in acell type, tissue, or organ.

A “fragment of a polynucleotide” is a nucleic acid that comprises all orany part of a given nucleotide molecule. An exemplary fragment is about6 kb in length, preferably having fewer nucleotides than about 6 kb,more preferably having fewer than about 1 kb.

“Reporter molecules” include chemical, radionucleic, enzymatic,fluorescent, chemiluminescent, or chromogenic agents which associatewith a particular nucleotide sequence, receptor, or amino acid sequence,thereby establishing the presence of or quantifying the expression of acertain sequence, receptor or agent binding to the receptor.

“Chimeric” oligonucleotides may be constructed by introducing all orpart of a nucleotide sequence of this invention into a vector containingadditional nucleic acid sequence which might be expected to change anyone or several of the following GPCR characteristics: cellular location,distribution, ligand-binding affinities, interchain affinities,degradation/turnover rate, signaling, etc. Similarly, chimeric peptidesare GPCR amino acid sequences which have been constructed to containadditional amino acid sequences which might be expected to change anyone or several of the following GPCR characteristics: cellular location,distribution, ligand-binding affinities, interchain affinities,degradation/turnover rate, signaling, etc.

“Active”, with respect to a GPCR, refers to those forms, fragments, ordomains of a GPCR polypeptide which retain the biological and/orantigenic activity of a GPCR polypeptide.

“Naturally occurring GPCR polypeptide” refers to a polypeptide producedby cells which are not genetically engineered and specificallycontemplates various polypeptides arising from post-translationalmodifications of the polypeptide including but not limited toacetylation, carboxylation, glycosylation, phosphorylation, lipidationand acylation.

“Derivative” refers to polypeptides which are chemically modified bytechniques such as ubiquitination, labeling, pegylation (derivatizationwith polyethylene glycol), insertion or substitution of amino acids suchas ornithine which do not normally occur in human proteins, or one ormore amino acids from the wild type sequence.

“Conservative amino acid substitutions” result from replacing one aminoacid with another having similar structural and/or chemical properties,such as the replacement of a leucine with an isoleucine or valine, anaspartate with a glutamate, or a threonine with a serine.

A “signal sequence” or “leader sequence” can be used, when desired, todirect the polypeptide through a membrane of a cell. Such a sequence maybe naturally present on polypeptides produced using the vectors andmethods of the present invention or provided from heterologous sourcesby recombinant DNA techniques.

“Inhibitor” is any substance which retards or prevents chemical orphysiological reactions or responses. Common inhibitors include but arenot limited to antisense molecules, antibodies, and antagonists.

“Standard expression” is a quantitative or qualitative measurement forcomparison. It is based on a statistically appropriate number of normalsamples and is created to use as a basis of comparison when performingdiagnostic assays, running clinical trials, or following patienttreatment profiles.

“Animal”, as used herein, includes human, domestic (e.g., cats, dogs,etc.), agricultural (e.g., cows, horses, sheep, etc.) or test species(e.g., mouse, rat, rabbit, etc.).

“Stringent conditions” refers to conditions that allow for thehybridization of essentially complementary nucleic acid sequences. Forinstance, such conditions will generally allow hybridization of sequencewith at least about 85% sequence identity, preferably with at leastabout 90% to 95% sequence identity, more preferably about 91% sequenceidentity, about 92% sequence identity, about 93% sequence identity,about 94% sequence identity, more preferably with at least about 95% to99% sequence identity, preferably about 96% sequence identity, about 97%sequence identity, about 98% sequence identity, still more preferablyabout 99% sequence identity, or about 100% sequence identity to thecomplementary nucleic acid sequences.

“Recombinant cells” encompasses one or more individual cells as well asto a recombinant cell line in which the cells are expressing aheterologous protein.

The term “extracellular signal” encompasses molecules and changes in thecellular environment that are transduced intracellularly via cellsurface proteins that interact, directly or indirectly, with theextracellular signal. An extracellular signal or effector moleculeincludes any compound or substance that in some manner alters theactivity of a cell surface protein. Examples of such signals include,but are not limited to, molecules such as acetylcholine, growth factorsand hormones, lipids, sugars and nucleotides that bind to cell surfacereceptors and modulate the activity of such receptors. The term,“extracellular signal” also includes as yet unidentified substances thatmodulate the activity of a cellular receptor, and thereby influenceintracellular functions. Such extracellular signals are potentialpharmacological agents that may be used to treat specific diseases bymodulating the activity of specific cell surface receptors.

The term “functional assays” as used herein encompasses those assaysthat take advantage of certain aspects of GPCR protein activity orbehavior under particular conditions, for example, the activation of a Gprotein upon binding of a ligand to the GPCR.

The term “selectively binds” as used herein refers to a compound (e.g.,an antibody, a peptide, a lipid or a small organic molecule) that bindsto a native polypeptide or to a chimeric polypeptide preferentiallyrelative to other unrelated polypeptides. A compound selectively bindsto the native polypeptide or a chimeric polypeptide of the invention ifit has at least a 10%, preferably at least a 25%, at least a 50%, atleast a 75%, at least a 90%, at least a 95%, or at least a 100% higheraffinity and/or avidity for the native polypeptide or chimericpolypeptide than an unrelated polypeptide.

Introduction

G protein coupled receptors (hereinafter termed “GPCRs”) comprise alarge superfamily of receptors. GPCRs were originally defined asreceptors that transduce signals from the extracellular compartment tothe interior through biochemical processes involving GTP-bindingproteins. Molecular cloning of the first receptor genes suggestedprotein structures with seven transmembrane α-helical domains (hence“7TM receptors”). Typical GPCRs do share a common structural motif ofseven transmembrane helical domains, but some GPCRs are insteadsingle-spanning transmembrane receptors for cytokines such aserythropoietin or insulin, or multi-polypeptide receptors such as thecollagen receptor.

As used herein, “GPCR protein” and “a GPCR” refers to a protein in whichone response to the binding of a ligand to the GPCR is the activation ofa G protein. This term also encompasses the amino acid and nucleotidesequences of these proteins. “A GPCR” is meant to include both thesingular and plural forms of the phrase, i.e., “a GPCR” may refer to oneor more GPCR molecules.

Modern crystallography and mutational analyses show that GPCRs areversatile receptors for a wide range of extracellular messengers,including biogenic amines, purines and nucleic acid derivatives, lipids,peptides and proteins, odorants, pheromones, tastants, ions like calciumand protons, and photons (in the case of rhodopsin). GPCRs can formhomo- and heterodimers, as well as complex receptosomes, which in somecases can incorporate additional intra- and extracellular soluble andtransmembrane proteins.

GPCRs play a vital role in the signaling processes that control cellularmetabolism, cell growth and motility, inflammation, neuronal signaling,and blood coagulation. G protein coupled receptor proteins also serve astargets for molecules such as hormones, neurotransmitters andphysiologically active substances. Thus, GPCRs are a major target fordrug action and development.

High Expression Vectors

In a first aspect, the invention provides a vector which facilitateshigh levels of expression of GPCR proteins in a cell line. Native GPCRproteins are expressed in levels numbering in the upper hundreds to lowthousands of copies per cell. Since most molecular and cell biologytechniques, such as screening assays and raising antibodies, cannot beconducted with such low levels of protein, high expression vectors areneeded to produce proteins on the order of tens of thousands to millionsof copies per cell. The vectors encompassed by the instant inventionovercome the hurdle to GPCR research posed by the low expression ofnative GPCR proteins by facilitating high levels of expression of GPCRproteins in mammalian cells.

In an exemplary embodiment, according to FIG. 10, the invention providesa novel vector named pMEX2 for expression of GPCR proteins on thecytoplasmic membrane of mammalian cells. As shown in FIG. 10, thisvector includes a pUC origin and a beta-lactamase gene for replicationand ampicillin selection of the plasmid in bacteria. A puromycinresistance marker for maintaining the plasmid in mammalian cells is alsoincluded in certain embodiments of this vector. Expression of the geneof interest is under control of a strong CMV promoter for high-leveltranscription activity.

In another exemplary embodiment, according to FIG. 12, the inventionprovides a novel vector named pMEX5 for expression of GPCR proteins onthe cytoplasmic membrane of mammalian cells. This vector allows forexpression of GPCR proteins under control of an inducible promoter. Asin pMEX2, pMEX5 includes a strong CMV promoter for high-leveltranscription activity, with the additional feature that the promoter isoperably linked to a tetracycline operator, as shown in FIG. 12. Inalternative embodiments of the invention, the inducible promoter may beselected from a chemical inducible promoter, such as asteroid-responsive promoter, a tissue responsive promoter, a promoterderived from the genome of mammalian cells, such as the metallothioneinpromoter, and a promoter derived from mammalian viruses, such as theretrovirus long terminal repeat, the adenovirus late promoter, and thevaccinia virus 7.5K promoter. Promoters produced by recombinant DNA orsynthetic techniques may also be used to provide for transcription of apolypeptide-encoding nucleotide sequence.

In both the vectors pictured in FIG. 10 and FIG. 12 respectively, thevectors include Kozak consensus sequence for optimal translationinitiation and an SV40 late polyadenylation signal to promote stabilityin the transcripts. Also included in the vectors are signal sequences(labeled “SP” in FIG. 10 and FIG. 12) which provide efficient deliveryof the translated protein to the membrane of the cell. It is intendedthat the exemplary signal sequences shown in FIG. 10 and FIG. 12 are notmeant to be limiting, and that the instant invention encompasses allpossibilities of signal sequences which are effective in targeting thetranslated protein to the cell membrane.

In an exemplary embodiment, the invention provides binding sites for thebacteriophage DNA binding protein LexA that are engineered just upstreamof the promoter sequence of the expression vector. An expression vectorengineered in this way will express a chimeric protein that includes theLexA DNA binding domain linked to an activation domain, such as that forthe herpes simplex virus protein VP16. In principle, this combination ofDNA binding site, DNA binding protein and activation domain can bemanipulated by using DNA binding sites, DNA binding proteins and/oractivation domains to strengthen the ability of the promoter to initiateand sustain transcription of downstream elements in the vector.

In one embodiment of the invention, the expression vector includes anucleotide sequence for a GPCR protein selected from one of severalpossible GPCR families of proteins, including: anaphylatoxin, apelin,bombesin, cannabinoid, chemokine, free fatty acid, galanin, glucagon,glycoprotein hormone, leukotriene/lipoxin, lysophospholipid,melanin-concentrating hormone, melatonin, N-formylpeptide, neuromedin U,neuropeptide S, neuropeptide W/neuropeptide B, neuropeptide Y, opioid,platelet activating factor, prolactin releasing peptide, prostanoid,PTH, purinergic, tachykinin, trace amine, and urotensin.

In another embodiment of the invention, the expression vector includes anucleotide sequence encoding for an “orphan” GPCR, which is a GPCRprotein for which there is as of yet no known ligand. There arecurrently over two hundred GPCR proteins identified as orphan GPCRs.These orphan proteins may be implicated in a number of disease, such ascancer and inflammation associated with arthritis, and thus orphan GPCRsare of particular interest to the pharmaceutical and biotechnologyindustries.

In a further embodiment of the invention, the expression vector includesa nucleotide sequence encoding for a GPCR that is a member selectedfrom: C3aR, APJ, BB1, BB3, GPR55, CCR1, CCR5, CCR7, CCR9, CMKLR1, CXCR3,CXCR4, FFA1, FFA2, GAL1, GAL2, GAL3, GHRH, TSH, ALX, BLT1, BLT2, CysLT1,LPA2, LPA3, MCH1, MT2, FPR1, NMU1, NPS, NPS(1), NPS(2), NPS Ile107,NPBW1, NPBW2, delta, kappa, mu, NOP, GPR37L1, GPR84, MRGX1, MRGX2, PSGR,PAF, PRP, DP, EP1, GPR44, PTH2, P2Y12, NK2, NK3, TA1, C5aR, PAR2.

In a still further embodiment, the expression vector includes a GPCRprotein which has an amino acid sequence that is a member selected from:SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, and SEQ ID NO: 17, aspictured in FIG. 1 through FIG. 9.

In a further embodiment of the invention, the expression vectorcomprises a member selected from: SEQ ID NO: 19 and SEQ ID NO: 20.Vector maps graphically illustrating the expression vectorscorresponding to these sequences are shown in FIG. 10 and FIG. 12respectively. The expression vectors in one embodiment of the inventioncontain multiple restriction sites which can be used to insert anucleotide sequence encoding for a GPCR.

In a further embodiment, the invention provides expression vectors thatinclude nucleotide sequences for molecules to aid in the detection ofGPCR. In a still further embodiment, such detection includes the abilityto determine if the protein is expressed in the correct orientation onthe cytoplasmic membrane of mammalian cells.

In a preferred embodiment, the invention provides expression vectorscontaining a pUC origin and a beta-lactamase gene for replication andampicillin selection of the plasmid in bacteria. In a further embodimentof the invention, the expression vector can also include a puromycinresistance marker for the gene of interest, which is under control of astrong CMV promoter for high-level transcription activity.

In a preferred embodiment of the invention, a method is provided forcreating expression vectors that enable expression of GPCRs, properlyfolded with appropriate post-translational modifications, with levels ofexpression of at least one million copies per cell on the cytoplasmicmembrane. This level of expression is suitable for whole-cellimmunization for raising antibodies as well as for functional andstructural studies of the receptor.

Kozak Sequence

Most eukaryotic mRNAs contain a short recognition sequence thatfacilitates the initial binding of mRNA to the small subunit of theribosome. The consensus sequence for initiation of translation invertebrates (also called Kozak sequence) is: ACCATG (see, e.g., SEQ IDNO: 19, position 1099-1104). More generally it is: GCCRCCATGG where R isa purine (A or G) (see, e.g., SEQ ID NO: 19, position 1096-1105). Toimprove expression levels, it may be advantageous to design the clonedinsert according to Kozak's rules in the present invention.

Recombinant Cells

In one aspect, the invention provides recombinant cell lines expressingGPCR proteins. In a preferred embodiment of the invention, expression ofGPCR proteins is governed by a high expression vector as describedabove. As used herein, the terms “recombinant cell line”, “cell line”,“recombinant cells” can be used interchangeably to refer to cellsheterologously expressing an indicated protein.

In one embodiment of the invention, the recombinant cells express levelsof GPCR of at least 150,000 protein molecules per cell. In a furtherembodiment of the invention, GPCR is expressed at a range of 200,000copies to 2,000,000 copies per cell. In a still further embodiment ofthe invention, the GPCR protein is expressed at a range of 400,000copies to 2,000,000 copies per cell. In a still further embodiment ofthe invention, the recombinant cells express GPCR protein at a range of600,000 copies to 2,000,000 copies per cell. In a yet further embodimentof the invention, the recombinant cells express GPCR protein at a rangeof 800,000 copies to 2,000,000 copies per cell. In a preferredembodiment of the invention, the recombinant cells express GPCR proteinat a range of 1,000,000 copies to 2,000,000 copies per cell. In anotherpreferred embodiment, the cells express GPCR protein at a range of1,500,000 copies to 2,000,000 copies per cell.

In yet another embodiment of the invention, the GPCR protein is derivedfrom an animal. In a further embodiment of the invention, the GPCRprotein is derived from a mammal, including rat, mouse or human.

In still another embodiment of the invention, the recombinant cellexpressing the GPCR protein is derived from a cell line, which may as anexample be selected from a Chinese hamster ovary (CHO) cell line, ahuman embryonic kidney cell line (HEK293T), a C6 glioma cell line, theRH7777 cell line, the SW480 cell line from human adenocarcinoma of thecolon, the VS35 cell line, the 1321N1 cell line, and other cell linesthat are known in the art to be amenable to stable or transienttransfection with heterologous nucleic acids.

In one aspect, the invention provides a method of producing a cell line,which includes creating at least one expression vector selected fromnucleotide sequence SEQ ID NO: 19 or SEQ ID NO: 20 and transfecting ahost cell with the expression vector. The transfection of the host cellcan either be such that it creates a stably transfected cell line or atransiently transfected cell line by methods known in the art.

In one embodiment of the invention, the stably or transientlytransfected cell line is a mammalian cell line. In a preferredembodiment, the cell line is derived from a cell line selected from agroup consisting of: CHO, HEK293T, C6, RH7777, SW480, VS35, and 1321N1.Mammalian cell lines are particularly preferred, because such cell linesensure that the protein will receive the proper post-translationalmodifications before being transported to the cell membrane.

GPCR-Expressing Recombinant Cells

In one embodiment, the invention provides recombinant cells expressing aGPCR protein that has an amino acid sequence selected from SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9. In a further embodiment ofthe invention, the GPCR protein is a member selected from C3aR (inaccordance with FIG. 6), APJ (in accordance with Accession numberNM_(—)005161), BB1 (in accordance with Accession number NM_(—)012799),BB3 (in accordance with Accession number NM_(—)001727), GPR55 (inaccordance with Accession number NM_(—)005683), CCR1 (in accordance withAccession number NM_(—)000579), CCR5 (in accordance with FIG. 7), CCR7(in accordance with Accession number NM_(—)001838), CCR9 (in accordancewith Accession number NM_(—)006641), CMKLR1 (in accordance withAccession number NM_(—)004072), CXCR3 (in accordance with Accessionnumber NM_(—)001504), CXCR4 (in accordance with FIG. 8), FFA1 (inaccordance with Accession number NM_(—)005303), FFA2 (in accordance withAccession number NM_(—)005304), GAL1 (in accordance with Accessionnumber NM_(—)001480), GAL2 (in accordance with Accession numberNM_(—)003857), GAL3 (in accordance with Accession number NM_(—)003614),GHRH (in accordance with Accession number NM_(—)000823), TSH (inaccordance with Accession number NM_(—)012888), ALX (in accordance withAccession number NM_(—)003857), BLT1 (in accordance with Accessionnumber BC_(—)004545), BLT2 (in accordance with Accession numberNM_(—)0193839.1), CysLT1 (in accordance with Accession numberNM_(—)006639), LPA2 (in accordance with Accession numberNM_(—)004724.4), LPA3 (in accordance with Accession numberNM_(—)012152.1), MCH1 (in accordance with Accession numberNM_(—)005297), MT2 (in accordance with Accession number NM_(—)005959),FPR1 (in accordance with Accession number NM_(—)002029), NMU1 (inaccordance with FIG. 2), NPS (in accordance with Accession numberNM_(—)175678), NPS(1) (in accordance with Accession numberNM_(—)207172), NPS(2) (in accordance with Accession numberNM_(—)207173), NPS Ile107 (in accordance with Accession numberSNP591694), NPBW1 (in accordance with Accession number NM_(—)001014784and NM_(—)005285), NPBW2 (in accordance with Accession numberNM_(—)005286), delta (in accordance with Accession number NM_(—)012617),kappa (in accordance with Accession number L22001), mu (in accordancewith Accession number L13069), NOP (in accordance with Accession numberBC038433), GPR37L1 (in accordance with Accession number NM_(—)004767),GPR84 (in accordance with Accession number NM_(—)020370), MRGX1 (inaccordance with Accession number NM_(—)147199), MRGX2 (in accordancewith Accession number NM_(—)054030), PSGR (in accordance with Accessionnumber NM_(—)030774), PAF (in accordance with Accession numberNM_(—)000952), PRP (in accordance with Accession number NM_(—)004248),DP (in accordance with Accession number NM_(—)000953), EP1 (inaccordance with Accession number NM_(—)000955), GPR44 (in accordancewith Accession number NM_(—)004778), PTH2 (in accordance with Accessionnumber NM_(—)005048), P2V 12 (in accordance with Accession numberNM_(—)022788), NK2 (in accordance with Accession number NM_(—)001057),NK3 (in accordance with Accession number NM_(—)175057), TA1 (inaccordance with Accession number NM_(—)138327), C5aR (in accordance withFIG. 1), PAR2 (in accordance with FIG. 9).

In another embodiment of the invention, recombinant cells express a GPCRprotein encoded by a nucleotide sequence selected from SEQ ID NO: 2, SEQID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 14, SEQID NO: 16, and SEQ ID NO: 18, as pictured in FIG. 1 through FIG. 9. In afurther embodiment of the invention, transcription of the nucleotidesequence encoding a GPCR protein is operably linked to a promoter, suchas the cytomegalovirus (CMV) promoter. In a still further embodiment ofthe invention, the promoter is itself operably linked to an inducibleoperator, for example a tetracycline operator.

In another embodiment of the invention, the recombinant cells expressGPCR protein at a level of at least 150,000 copies per cell. In afurther embodiment, the invention provides GPCR expression levels at arange of about 150,000 copies and 2,000,000 copies per cell. In a stillfurther embodiment, the invention provides GPCR expression levels at arange of about 200,000 copies and 2,000,000 copies per cell. In a stillfurther embodiment, the invention provides GPCR expression levels at arange of about 300,000 copies and 2,000,000 copies per cell. In afurther embodiment, the invention provides GPCR expression levels at arange of about 400,000 copies and 2,000,000 copies per cell. In a stillfurther embodiment, the invention provides GPCR expression levels at arange of about 500,000 copies and 2,000,000 copies per cell. In a stillfurther embodiment, the invention provides GPCR expression levels at arange of about 600,000 copies and 2,000,000 copies per cell. In a stillfurther embodiment, the invention provides GPCR expression levels at arange of about 700,000 copies and 2,000,000 copies per cell. In a stillfurther embodiment, the invention provides GPCR expression levels at arange of about 800,000 copies and 2,000,000 copies per cell. In a stillfurther embodiment, the invention provides GPCR expression levels at arange of about 900,000 copies and 2,000,000 copies per cell. In a stillfurther embodiment, the invention provides GPCR expression levels at arange of about 1,000,000 copies and 2,000,000 copies per cell. In astill further embodiment, the invention provides GPCR expression levelsat a range of about 1,500,000 copies and 2,000,000 copies per cell. In astill further embodiment, the invention provides GPCR expression levelsat a range of about 1,750,000 copies and 2,000,000 copies per cell.

In one aspect, the invention provides a recombinant cell line stablyexpressing a GPCR protein that has an amino acid sequence selected fromSEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, and SEQ ID NO: 17. It will beunderstood by those of skill in the art that these amino acid sequencesencompass the nucleic acid sequences which encode for them.

Nucleotide and Amino Acid Sequences Encoding for GPCR

In preferred embodiments, the instant invention uses nucleotide andamino acid sequences encoding for GPCR proteins in functional andcell-based assays and to produce recombinant cell lines. The nucleotidesequences encoding GPCRs (or their complements) have numerousapplications in techniques known to those skilled in the art ofmolecular biology. These techniques include their use: as hybridizationprobes, in the construction of oligomers for PCR, for chromosome andgene mapping, in the recombinant production of GPCR, and in generationof antisense DNA or RNA, their chemical analogs and the like. Uses ofnucleotides encoding a GPCR disclosed herein are exemplary of knowntechniques and are not intended to limit their use in any techniqueknown to a person of ordinary skill in the art. Furthermore, thenucleotide sequences disclosed herein may be used in molecular biologytechniques that have not yet been developed, provided the new techniquesrely on properties of nucleotide sequences that are currently known,e.g., the triplet genetic code, specific base pair interactions, etc.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of GPCR-encodingnucleotide sequences may be produced. Some of these will bear onlyminimal homology to the nucleotide sequence of the known and naturallyoccurring GPCR. The invention has specifically contemplated each andevery possible variation of nucleotide sequence that could be made byselecting combinations based on possible codon choices. Thesecombinations are made in accordance with the standard triplet geneticcode as applied to the nucleotide sequence of naturally occurring GPCR,and all such variations are to be considered as being specificallydisclosed.

Although the nucleotide sequences which encode a GPCR, its derivativesor its variants are preferably capable of hybridizing to the nucleotidesequence of the naturally occurring GPCR polynucleotide under stringentconditions, it may be advantageous to produce nucleotide sequencesencoding GPCR polypeptides or their derivatives which posses asubstantially different codon usage. Codons can be selected to increasethe rate at which expression of the peptide occurs in a particularprokaryotic or eukaryotic expression host in accordance with thefrequency with which particular codons are utilized by the host. Otherreasons for substantially altering the nucleotide sequence withoutaltering the encoded amino acid sequence include the production of RNAtranscripts with certain desirable properties, such as an increasedhalf-life or greater specificity than is possible with the naturallyoccurring sequence.

Nucleotide sequences encoding GPCR polypeptides may be joined to avariety of other nucleotide sequences by means of well establishedrecombinant DNA techniques. Useful nucleotide sequences for joining toGPCR-encoding polynucleotides include an assortment of cloning vectorssuch as plasmids, cosmids, lambda phage derivatives, phagemids, and thelike. Vectors of interest include expression vectors, replicationvectors, probe generation vectors, sequencing vectors, etc. In general,vectors of interest may contain an origin of replication functional inat least one organism, convenient restriction endonuclease sensitivesites, and selectable markers for one or more host cell systems.

It will be recognized that many deletional or mutational analogs of GPCRpolynucleotides will be effective hybridization probes for GPCRpolynucleotides. Accordingly, the invention relates to nucleic acidsequences that hybridize with such GPCR encoding nucleic acid sequencesunder stringent conditions.

Hybridization conditions and probes can be adjusted inwell-characterized ways to achieve selective hybridization ofhuman-derived probes. Examplary stringent conditions, include a buffercontaining 1 mM EDTA, 0.5 M NaHPO₄ (pH 7.2), 7% (w/v) SDS.

Nucleic acid molecules that will hybridize to GPCR polynucleotides understringent conditions can be identified functionally. Without limitation,examples of hybridization probes include probes and primers used foridentifying tissues that express GPCR, measuring mRNA levels, forinstance to identity a sample's tissue type or to identify cells thatexpress abnormal levels of GPCR, and detecting polymorphisms of GPCR.

It is possible to produce a DNA sequence, or portions thereof, entirelyby synthetic chemistry. After synthesis, the nucleic acid sequence canbe inserted into any of the many available DNA vectors and theirrespective host cells using techniques known in the art. Moreover,synthetic chemistry may be used to introduce mutations into a nucleotidesequence. A portion of sequence in which a mutation is desired can alsobe synthesized and recombined with longer portion of an existing genomicor recombinant sequence.

GPCR polynucleotides may be used to produce a purified oligo- orpolypeptide using well known methods of recombinant DNA technology. Theoligopeptide may be expressed in a variety of host cells, eitherprokaryotic or eukaryotic. Host cells may be from the same species fromwhich the nucleotide sequence was derived or from a different species.Advantages of producing an oligonucleotide by recombinant DNA technologyinclude obtaining adequate amounts of the protein for purification andthe availability of simplified purification procedures.

Sequences encoding GPCR can be synthesized, in whole or in part, usingchemical methods well known in the art. Alternatively, GPCR itself canbe produced using chemical methods to synthesize its amino acidsequence, such as by direct peptide synthesis using solid-phasetechniques. Protein synthesis can either be performed using manualtechniques or by automation. Automated synthesis can be achieved, forexample, using Applied Biosystems 431A Peptide Synthesizer (PerkinElmer). Optionally, fragments of GPCR can be separately synthesized andcombined using chemical methods to produce a full-length molecule.

The newly synthesized peptide can be substantially purified bypreparative high performance liquid chromatography. The composition of asynthetic GPCR can be confirmed by amino acid analysis or sequencing.Additionally, any portion of the amino acid sequence of GPCR can bealtered during direct synthesis and/or combined using chemical methodswith sequences from other proteins to produce a variant polypeptide or afusion protein.

As will be understood by those of skill in the art, it may beadvantageous to produce GPCR polynucleotides possessing non-naturallyoccurring codons. For example, codons preferred by a particularprokaryotic or eukaryotic host can be selected to increase the rate ofprotein expression or to produce an RNA transcript having desirableproperties, such as a half-life which is longer than that of atranscript generated from the naturally occurring sequence.

The nucleotide sequences referred to herein can be engineered usingmethods generally known in the art to alter GPCR polynucleotides for avariety of reasons, including but not limited to, alterations whichmodify the cloning, processing, and/or expression of the polypeptide ormRNA product. DNA shuffling by random fragmentation and PCR reassemblyof gene fragments and synthetic oligonucleotides can be used to engineerthe nucleotide sequences. For example, site-directed mutagenesis can beused to insert new restriction sites, alter glycosylation patterns,change codon preference, produce splice variants, introduce mutations,and so forth.

Similarly, the polypeptide sequences referred to herein can beengineered or modified, preferably with post-translational modificationthat occur in the naturally occurring polypeptides, such asglycosylation.

Antibodies

In one aspect, the invention provides an antibody or antigen bindingfragment that specifically binds to a structural feature of a GPCRprotein. Such an antibody or antigen binding fragment is raised againstan immunogen, which in a further aspect of the invention is a cell lineexpressing between 150,000 and 2,000,000 copies of GPCR protein percell. In one embodiment of the invention, the antibody or antigenbinding fragment is a monoclonal antibody or antigen binding fragment.

In one embodiment of the invention, the antibody or antigen bindingfragment binds to a structural feature of a GPCR protein which is amember of a GPCR family selected from anaphylatoxin, apelin, bombesin,cannabinoid, chemokine, free fatty acid, galanin, glucagon, glycoproteinhormone, leukotriene/lipoxin, lysophospholipid, melanin-concentratinghormone, melatonin, N-formylpeptide, neuromedin U, neuropeptide S,neuropeptide W/neuropeptide B, neuropeptide Y, opioid, plateletactivating factor, prolactin releasing peptide, prostanoid, PTH,purinergic, tachykinin, trace amine, and urotensin.

In a further embodiment, the invention provides an antibody or antigenbinding fragment which is raised against a cell line expressing a memberof a GPCR-family selected from C3aR, APJ, BB1, BB3, GPR55, CCR1, CCR5,CCR7, CCR9, CMKLR1, CXCR3, CXCR4, FFA1, FFA2, GAL1, GAL2, GAL3, GHRH,TSH, ALX, BLT1, BLT2, CysLT1, LPA2, LPA3, MCH1, MT2, FPR1, NMU1, NPS,NPS(1), NPS(2), NPS Ile107, NPBW1, NPBW2, delta, kappa, mu, NOP,GPR37L1, GPR84, MRGX1, MRGX2, PSGR, PAF, PRP, DP, EP1, GPR44, PTH2,P2Y12, NK2, NK3, TA1, C5aR, and PAR2. In a still further embodiment, theantibody or antigen binding fragment recognizes an epitope on a GPCRprotein selected from C3aR, APJ, BB1, BB3, GPR55, CCR1, CCR5, CCR7,CCR9, CMKLR1, CXCR3, CXCR4, FFA1, FFA2, GAL 1, GAL2, GAL3, GHRH, TSH,ALX, BLT1, BLT2, CysLT1, LPA2, LPA3, MCH1, MT2, FPR1, NMU1, NPS, NPS(1),NPS(2), NPS Ile107, NPBW1, NPBW2, delta, kappa, mu, NOP, GPR37L1, GPR84,MRGX1, MRGX2, PSGR, PAF, PRP, DP, EP1, GPR44, PTH2, P2Y12, NK2, NK3,TA1, C5aR, and PAR2.

In another embodiment, the invention provides an antibody or antigenbinding fragment which is raised against a cell line expressing a memberof GPCR family having an amino acid sequence selected from SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,SEQ ID NO: 13, SEQ ID NO: 15, and SEQ ID NO: 17.

In a further embodiment, the invention provides an antibody or antigenbinding fragment that binds to a structural feature of a GPCR proteinand is raised against an immunogen which is a cell line expressing atleast about 150,000 copies of a GPCR protein per cell. In a preferredembodiment, the cell line expresses about 500,000 copies of a GPCRprotein per cell. In a particularly preferred embodiment, the cell lineexpresses between about 1,000,000 and about 2,000,000 copies of a GPCRprotein per cell.

In one aspect, the invention provides a method for producing monoclonalantibodies for a GPCR protein. This method includes immunizing a testanimal with at least one cell line expressing a member of a GPCR family.In accordance with the invention, this cell line expresses at leastabout 150,000 copies of said GPCR protein per cell. The test animal isinduced to produce hybridomas, which are isolated. The method alsoincludes screening for monoclonal antibodies using one or morecell-based assay systems. In one embodiment of the invention, thecell-based assay systems used to screen for monoclonal antibodies areselected from a group consisting of: FACS, ELISA, calcium imaging,FLIPR, multiplex ligand binding, and electrophysiology.

In one embodiment of the invention, monoclonal antibodies are induced ina test animal selected from a group comprising: rabbit, mouse, rat, pig,dog, monkey and goat. In a preferred embodiment, the invention providesa method of immunizing the test animal with whole cells expressing saidmember of GPCR family. In accordance with the invention, the whole cellsused to immunize the test animal express at least 150,000 copies of themember of the GPCR family per cell.

GPCRs have traditionally been good drug targets for small moleculecompounds and peptides. The field of antibody therapeutics for GPCRs isstill in early-stage clinical trials. However, cases where small ligandscan not be obtained, such as for example, Family II GPCRs, monoclonalantibodies can provide a viable alternative approach. Monoclonalantibodies may bind and lock GPCR in its active form and function asagonists. In addition, since the extracellular domains of GPCRs are morediverse than the rest of GPCR proteins including the transmembranedomains that small molecule compounds typically bind, monoclonalantibodies may bind to GPCRs more specifically than small molecules andthus can better distinguish subtle sequence and structural differenceswithin sub-family members. Since GPCRs are also known to beoverexpressed in many tumors, an advantage of GPCR antibody therapeuticsis their ability to act as targeting moieties, guiding specific andaccurate destruction of cancer cells.

Kits

In one aspect, the invention provides a kit for high throughputpurification and quantification of a plurality of recombinant proteinsof one or more members of GPCR family. The kit includes a vector forexpressing said recombinant proteins in host cells, wherein said vectorcomprises SEQ ID NO: 19 or 20, an affinity chromatography resin, aproteolytic enzyme, an internal quantification standard, a matrix forMALDI-TOF mass spectrometry, and instructions for use. In oneembodiment, the invention further provides a kit that also includes atleast one buffer selected from the group consisting of a lysis buffer; adenaturing buffer; an affinity chromatography binding buffer; anaffinity chromatography washing buffer; an affinity chromatographyelution buffer; and a proteolytic digestion buffer.

In another embodiment, the invention provides a kit for high throughputpurification and quantification that includes at least one multi-wellplate. In yet another embodiment, the invention provides a kit for highthroughput purification and quantification which includes a partially orfully automated high throughput purification and quantification system.

In a further embodiment, the invention provides a kit which includes avector that induces expression of one or more members of one or moreGPCR families at a level of at least about 150,000 copies per cell. In apreferred embodiment, the vector induces between 150,000 and 2,000,000copies of the GPCR protein per cell.

Screening Methods

In one aspect, the invention provides methods for screening fortherapeutic candidates. These methods include the use of a recombinantcell expressing a GPCR protein, where a test entity is contacted withthe recombinant cell, and binding is detected between the test entityand the GPCR protein. As an embodiment of the invention, specificbinding activity of the test entity to the GPCR protein identifies thattest entity as a therapeutic candidate. The recombinant cell in thismethod expresses at least about 150,000 copies of the GPCR protein percell. In a further embodiment of the invention, the test entity iscontacted with a membrane extract of said recombinant cell. In apreferred embodiment of the invention, the method of screening for atherapeutic candidate employs a high throughput screen for detectingbinding of the test entity to the GPCR protein. In a particularlypreferred embodiment, the high throughput screen is partially or fullyautomated.

In one embodiment, the method for screening for therapeutic candidatesincludes a detection of binding of the test entity to the GPCR proteinby means of a fluorescent, chemical, radiological, or enzymatic reportermolecule.

In an embodiment of the invention, the therapeutic candidate is screenedfor the treatment of cancer or of an illness associated withinflammation. In a preferred embodiment of the invention, thetherapeutic candidate is screened for treatment of breast cancer.

In one aspect, the invention provides a method for identifying DNAsequences encoding a member of a GPCR family, which includes probing acDNA library or a genomic library with a labeled probe with a nucleotidesequence selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, and 18.DNA sequences from the library that are able to hybridize to the probeunder stringent conditions are thus identified as encoding a member of aGPCR family. In an embodiment of the invention, the cDNA library orgenomic library is derived from human tissue. In a particularlypreferred embodiment, the human tissue used to create the cDNA orgenomic library includes cancerous cells.

In one embodiment, the invention provides methods of using recombinantcell lines expressing the vectors of the present invention to preparecDNA libraries of GPCRs. Such high expressing cells will be rich inGPCRs, which can in one embodiment of the invention be screened bylow-stringency hybridization, or, alternatively, used in a polymerasechain reaction for amplification of candidate genes using degeneratepolymers. Proof-of-function can obtained after the expression of thecloned receptor in heterologous cells with an elicited agonist response.

The compounds tested as modulators of GPCRs can be any small chemicalcompound, or a biological entity, e.g., a macromolecule such as aprotein, sugar, nucleic acid or lipid. Alternatively, modulators can begenetically altered versions of GPCR. Typically, test compounds will besmall chemical molecules and peptides. Essentially any chemical compoundcan be used as a potential modulator or ligand in the assays of theinvention, although most often compounds can be dissolved in aqueous ororganic (especially DMSO-based) solutions are used. The assays aredesigned to screen large chemical libraries by automating the assaysteps and providing compounds from any convenient source to assays,which are typically run in parallel (e.g., in microtiter formats onmicrotiter plates in robotic assays). It will be appreciated that thereare many suppliers of chemical compounds, including Sigma (St. Louis,Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), FlukaChemika-Biochemica Analytika (Buchs Switzerland) and the like.

Blinding Assays

The instant invention provides binding assays using recombinant celllines claimed and described herein. Candidate or test compounds oragents which bind to GPCR and/or have a stimulatory or inhibitory effecton the activity or the expression of GPCR are identified either inassays that employ cells which express GPCR on the cell surface(cell-based assays) or in assays with isolated GPCR (cell-free assays).The various assays can employ a variety of variants of GPCR (e.g.,full-length GPCR, a biologically active fragment of GPCR, or a fusionprotein which includes all or a portion of GPCR). Moreover, GPCR can bederived from any suitable mammalian species (e.g., human GPCR, rat GPCRor murine GPCR). The assay can be a binding assay entailing direct orindirect measurement of the binding of a test compound or a known GPCRligand to GPCR. The assay can also be an activity assay entailing director indirect measurement of the activity of GPCR.

In one embodiment, the invention provides assays for screening candidateor test compounds which bind to or modulate the activity of amembrane-bound (cell surface expressed) form of GPCR. Such assays canemploy full-length GPCR, a biologically active fragment of GPCR, or afusion protein which includes all or a portion of GPCR. Such testcompounds can be obtained by any suitable means, e.g., from conventionalcompound libraries. Determining the ability of the test compound to bindto a membrane-bound form of GPCR can be accomplished, for example, bycoupling the test compound with a radioisotope or enzymatic label suchthat binding of the test compound to the GPCR expressing cell can bemeasured by detecting the labeled compound in a complex. For example,the test compound can be labeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H, eitherdirectly or indirectly, and the radioisotope detected by-direct countingof radio-emission or by scintillation counting. Alternatively, the testcompound can be enzymatically labeled with, for example, horseradishperoxidase, alkaline phosphatase, or luciferase, and the enzymatic labeldetected by determination of conversion of an appropriate substrate toproduct.

Binding assays can also be used to detect receptor-mediated G-proteinactivation (see, e.g., “Regulation of G Protein-Coupled ReceptorFunction and Expression” ed. Benovic, J. L. pp 119 132, 2000,Wiley-Liss, New York). Such assays include receptor-stimulated GTPBinding to G.alpha. subunits. Activation of GPCR results in GDP-GTPexchange in the G.alpha. subunit, and this exchange can be quantifiedand used as a direct measurement of receptor-G protein interaction. Thistypically involves the use of radiolabelled guanine nucleotide with thereceptor either in cell free membrane preparations or artificial lipidmembranes. The amount of radiolabel incorporated is used as a measure ofthe extent of G protein activation.

Receptor-G-protein interactions can also be examined. For example, inthe absence of GTP, an activator will lead to the formation of a tightcomplex of a G protein (all three subunits) with the receptor. Thiscomplex can be detected in a variety of ways, as noted above. Such anassay can be modified to search for inhibitors. Adding an activator tothe receptor and G protein in the absence of GTP, can be used to screenfor inhibitors through measurements of the dissociation constants of thereceptor-G protein complex. In the presence of GTP, release of the alphasubunit of the G protein from the other two G protein subunits serves asa criterion of activation.

Ligand binding to GPCR, a domain of a GPCR protein, or a chimericprotein can be tested in solution, in a bilayer membrane, attached to asolid phase, in a lipid monolayer, or in vesicles. Binding of amodulator can be tested using, e.g., changes in spectroscopiccharacteristics (e.g., fluorescence, absorbance, refractive index)hydrodynamic (e.g., shape), chromatographic, or solubility properties,as well as other techniques known in the art.

Other useful binding assays utilize changes in intrinsic tryptophanfluorescence of protein subunits. The intrinsic fluorescence oftryptophan residues undergoes an enhancement during GDP-GTP exchange.Such an enhancement can be detected using methods known in the art.

The assay can also be an expression assay entailing direct or indirectmeasurement of the expression of GPCR mRNA or GPCR protein. The variousscreening assays are combined with an in vivo assay entailing measuringthe effect of the test compound on the symptoms of hematological andcardiovascular diseases, disorders of the peripheral and central nervoussystem, COPD, asthma, genito-urological disorders and inflammationdiseases.

In a competitive binding format, binding assays comprise contacting GPCRexpressing cell with a known compound which binds to GPCR to form anassay mixture, contacting the assay mixture with a test compound, anddetermining the ability of the test compound to interact with the GPCRexpressing cell, wherein determining the ability of the test compound tointeract with the GPCR expressing cell comprises determining the abilityof the test compound to preferentially bind the GPCR expressing cell ascompared to the known compound.

In another embodiment, the assay is a cell-based assay comprisingcontacting a cell expressing a membrane-bound form of GPCR (e.g.,full-length GPCR, a biologically active fragment of GPCR, or a fusionprotein which includes all or a portion of GPCR) expressed on the cellsurface with a test compound and determining the ability of the testcompound to modulate (e.g., stimulate or inhibit) the activity of themembrane-bound form of GPCR. Determining the ability of the testcompound to modulate the activity of the membrane-bound form of GPCR canbe accomplished by any method suitable for measuring the activity of aG-protein coupled receptor or other seven-transmembrane receptors. Theactivity of a seven-transmembrane receptors can be measured in a numberof ways, not all of which are suitable for any given receptor. Among themeasures of activity are: alteration in intracellular Ca.sup.2+concentration, activation of phospholipase C, alteration inintracellular inositol triphosphate QP3) concentration, alteration inintracellular diacylglycerol (DAG) concentration, and alteration inintracellular adenosine cyclic 3′,5′-monophosphate (cAMP) concentration.

The cell-free assays of the present invention are amenable to use witheither a membrane-bound form of a GPCR or a soluble fragment thereof. Inthe case of cell-free assays comprising the membrane-bound form of thepolypeptide, it may be desirable to utilize a solubilizing agent suchthat the membrane-bound form of the polypeptide is maintained insolution. Examples of such solubilizing agents include but are notlimited to non-ionic detergents such as n-octylglucoside,n-dodecyl-glucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide,decanoyl-N-methyl-glucamide, Triton X-100, Triton X-114, Thesit,Isotridecypoly(ethylene glycol ether)n,3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CRAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

In some embodiments of the assays used in accordance with the presentinvention, it may be desirable to immobilize GPCR (or a GPCR targetmolecule) to facilitate separation of complexed from uncomplexed formsof one or both of the proteins, as well as to accommodate automation ofthe assay. Binding of a test compound to GPCR, or interaction of GPCRwith a target molecule in the presence and absence of a candidatecompound, can be accomplished in any vessel suitable for containing thereactants. Examples of such vessels include microtitre plates, testtubes, and micro-centrifuge tubes. In one embodiment, a fusion proteincan be provided which adds a domain that allows one or both of theproteins to be bound to a matrix. For example, glutathione-S-transferase(GST) fusion proteins or glutathione-S-transferase fusion proteins canbe adsorbed onto glutathione sepharose beads (Sigma Chemical; St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with the test compound or the test compound and either thenon-adsorbed target protein or GPCR, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotitre plate wells are washed to remove any unbound components andcomplex formation is measured either directly or indirectly, forexample, as described above. Alternatively, the complexes can bedissociated from the matrix, and the level of binding or activity ofGPCR can be determined using standard techniques.

In binding assays, either the test compound or the GPCR polypeptide cancomprise a detectable label, such as a fluorescent, radioisotopic,chemiluminescent, or enzymatic-label, such as horseradish peroxidase,alkaline phosphatase, or luciferase. Detection of a test compound whichis bound to GPCR polypeptide can then be accomplished, for example, bydirect counting of radioemmission, by scintillation counting, or bydetermining conversion of an appropriate substrate to a detectableproduct. Alternatively, binding of a test compound to a GPCR polypeptidecan be determined without labeling either of the interactants. Forexample, a microphysiometer can be used to detect binding of a testcompound with a GPCR polypeptide. A microphysiometer (e.g., Cytosensor™)is an analytical instrument that measures the rate at which a cellacidifies its environment using a light-addressable potentiometricsensor (LAPS). Changes in this acidification rate can be used as anindicator of the interaction between a test compound and GPCR [Haseloff,(1988)].

In another embodiment of the invention, a GPCR-like polypeptide can beused as a “bait protein” in a two-hybrid assay or three-hybrid assay[Szabo, (1995); U.S. Pat. No. 5,283,317), to identify other proteinswhich bind to or interact with GPCR and modulate its activity. Methodsfor detecting such complexes, in addition to those described above forthe GST-immobilized complexes, include immunodetection of complexesusing antibodies which specifically bind to GPCR polypeptide or testcompound, enzyme-linked assays which rely on detecting an activity ofGPCR polypeptide, and SDS gel electrophoresis under non-reducingconditions.

Functional Assays

In one aspect, the invention provides methods for producing functionalassay cell lines. These methods include producing cell lines expressingGPCR proteins encoded for by nucleotide sequences selected from SEQ IDNO: 2, 4, 6, 8, 10, 12, 14, 16, and 18. A functional reporter is coupledto the binding of a ligand to the GPCR protein, such that a bindingevent between said ligand and said GPCR protein is detectable as areporter activity readout. In a preferred embodiment of the invention,the cell line expressing GPCR proteins is expressing at levels of atleast 150,000 copies of GPCR protein per cell.

In one embodiment, the reporter readout that is detectable upon bindingof a ligand to the GPCR protein includes detection of second messengeractivity. In a preferred embodiment, the second messenger activityincludes a change in intracellular calcium levels, cAMP activity, and/orNFAT or CRE driven beta-lactamase.

In another embodiment, the reporter activity readout includes detectionof GFP, luciferase, and of a radio-labeled molecule.

In a second aspect, the invention provides a method of using aGPCR-expressing cell line to identify a test compound which modulatesactivity of said GPCR. In this method, second messenger activity ismeasured in a cell line in the absence of said test compound—thisconstitutes the first measurement. A second measurement is made ofsecond messenger activity in the presence of a test compound. Acomparison of the first and second measurement that shows that there isa difference between the first measurement and the second measurementidentifies the test compound as an agent modulates the activity of theGPCR. In accordance with the invention, the cell line expresses at least150,000 copies of the GPCR protein per cell.

In a preferred embodiment, the second messenger activity measured todetermine if a test compound modulates GPCR activity includes a changein intracellular calcium and a change in intracellular cAMP levels. Inanother preferred embodiment, the detection of second messenger activityincludes high throughput screening methods.

In one embodiment of the invention, an affinity tag is attached to aGPCR protein, allowing for detection of the protein when expressed onthe cellular membrane. With such an affinity tag, fluorescence-activatedcell sorting (FACS) can be used for both detection as well as toquantify protein expression. In a further embodiment of the invention,FACS screening makes use of an anti-tag monoclonal antibody for bothdetection and quantification. In another embodiment of the invention,recombinant receptors are similarly analyzed using radio-labeled ligandscombined with binding assays.

Flow cytometry is a method that can be utilized to detect surfaceexpression of GPCRs. In traditional flow cytometry, it is common toanalyze very large numbers of eukaryotic cells in a short period oftime. Newly developed flow cytometers can analyze and sort up to 20,000cells per second. In a typical flow cytometer, individual particles passthrough an illumination zone and appropriate detectors, gatedelectronically, measure the magnitude of a pulse representing the extentof light scattered. The magnitude of these pulses are sortedelectronically into “bins” or “channels”, permitting the display ofhistograms of the number of cells possessing a certain quantitativeproperty as a function of channel number (Davey and Kell, 1996). It hasbeen shown that the data accruing from flow cytometric measurements canbe analyzed (electronically) rapidly enough that electronic cell-sortingprocedures could be used to sort cells with desired properties intoseparate “buckets”, a procedure usually known as fluorescence-activatedcell sorting (Davey and Kell, 1996).

Fluorescence-activated cell sorting (FACS) is often used in studies ofhuman and animal cell lines and the control of cell culture processes.Fluorophore labeling of cells and measurement of the fluorescence canprovide quantitative data about specific target molecules or subcellularcomponents and their distribution in the cell population. Flow cytometrycan quantitate virtually any cell-associated property or cell organellefor which there is a fluorescent probe (or natural fluorescence). Theparameters which can be measured have previously been of particularinterest in animal cell culture.

FACS machines have been employed in the present invention to analyze thesuccess of various expression vectors and recombinant cell lines inproducing high levels of GPCR proteins. Detection and countingcapabilities of the FACS system are also encompassed in the methods ofthe present invention.

Measuring Intracellular Calcium Levels

In one embodiment of the invention, measurement of intracellular calciumlevels provides an indication of second messenger activity. Methods ofmeasuring intracellular calcium are known to those of skill in the art.For instance, a commonly used technique is the expression of receptorsof interest in Xenopus laevis oocytes followed by measurement of calciumactivated chloride currents (see Weber, 1999, Biochim Biophys Acta1421:213 233). In addition, several calcium sensitive dyes are availablefor the measurement of intracellular calcium. Such dyes can be membranepermeant or non-membrane permeant. Examples of useful membrane permeantdyes include acetoxymethyl ester forms of dyes that can be cleaved byintracellular esterases to form a free acid, which is no longer membranepermeant and remains trapped inside a cell. Dyes that are non-membranepermeant can be introduced into the cell by microinjection, chemicalpermeabilization, scrape loading and similar techniques (Haughland,1993, in “Fluorescent and Luminescent Probes for Biological Activity”ed. Mason, W. T. pp 34 43; Academic Press, London; Haughland, 1996, in“Handbook of Fluorescent Probes and Research Chemicals”, sixth edition,Molecular Probes, Eugene, Oreg.).

Included in the present invention are assays designed to directlymeasure levels of cAMP produced upon modulation of adenylate cyclaseactivity by GPCRs. Such assays are based on the competition betweenendogenous cAMP and exogenously added biotynilated cAMP. The capture ofcAMP is achieved by using a specific antibody conjugated to a solidmaterial such as capture beads. Such assays are efficient at measuringboth agonist and antagonist activities.

Other assays can involve determining the activity of receptors which,when activated, result in a change in the level of intracellular cyclicnucleotides, e.g., cAMP or cGMP, by activating or inhibiting downstreameffectors such as adenylate cyclase. There are cyclic nucleotide-gatedion channels, e.g., rod photoreceptor cell channels and olfactory neuronchannels that are permeable to cations upon activation by binding ofcAMP or cGMP (see, e.g., Altenhofen et al., Proc. Natl. Acad. Sci.U.S.A. 88:9868-9872 (1991) and Dhallan et al., Nature 347:184-187(1990)). In cases where activation of the receptor results in a decreasein cyclic nucleotide levels, it may be preferable to expose the cells toagents that increase intracellular cyclic nucleotide levels, e.g.,forskolin, prior to adding a receptor-activating compound to the cellsin the assay. Cells for this type of assay can be made byco-transfection of a host cell with DNA encoding a cyclicnucleotide-gated ion channel, GPCR phosphatase and DNA encoding areceptor (e.g., certain glutamate receptors, muscarinic acetylcholinereceptors, dopamine receptors, serotonin receptors, and the like),which, when activated, causes a change in cyclic nucleotide levels inthe cytoplasm. In one embodiment, changes in intracellular cAMP or cGMPcan be measured using immunoassays.

Other screening techniques include the use of cells which express GPCR(for example, transfected CHO cells) in a system which measuresextracellular pH changes caused by receptor activation [Iwabuchi,(1993)].

Functional Assay Panels

In one aspect, the present invention provides a series of functionalassay panels for use in screening for modulators of GPCRs for variousapplications, such as for therapeutics for treatment and diagnosis ofillnesses associated with GPCR activity.

The functional assay panels of the present invention are based on stablecell lines expressing different classes of GPCR. These cell lines areused as a source of representative targets for surveying drug candidatespecificity in functional assays such as calcium influx. These assaypanels typically include forty to fifty different GPCR-expressing celllines, but can include upwards of 300 cell lines. In addition, thesepanels can be customized to screen particular compounds against GPCRexpressing cell lines, such as those cell lines expressing GPCRs knownto be involved in certain illnesses.

In one embodiment, samples are assigned a relative GPCR activity valueof 100. Inhibition of GPCR is achieved when the GPCR activity valuerelative to control is about 90%, optionally 50%, optionally 25-0%.Activation of an GPCR is achieved when the GPCR activity value relativeto the control is 110%, optionally 150%, 200-500%, or 1000-2000%.

Assays for mRNA

In one embodiment of the invention, transcription levels can be measuredto assess the effects of a test compound on signal transduction. A hostcell containing the protein of interest is contacted with a testcompound for a sufficient time to effect any interactions, and then thelevel of gene expression is measured. The amount of time to effect suchinteractions may be empirically determined, such as by measuring thelevel of transcription as a function of time. The amount oftranscription may be measured by using methods known to those of skillin the art. For example, mRNA expression of the protein of interest maybe detected using Northern blots or their polypeptide products may beidentified using immunoassays. Alternatively, transcription based assaysusing reporter gene may be used as described in U.S. Pat. No. 5,436,128,herein incorporated by reference. The reporter genes can be, e.g.,chloramphenicol acetyltransferase, firefly luciferase, bacterialluciferase, .beta.-galactosidase and alkaline phosphatase. Furthermore,the protein of interest can be used as an indirect reporter viaattachment to a second reporter such as green fluorescent protein (see,e.g., Mistili & Spector, Nature Biotechnology 15:961-964 (1997)).

The amount of transcription is then compared to the amount oftranscription in either the same cell in the absence of the testcompound, or it may be compared with the amount of transcription in asubstantially identical cell that lacks the protein of interest. Asubstantially identical cell may be derived from the same cells fromwhich the recombinant cell was prepared but which had not been modifiedby introduction of heterologous DNA.

Solid State and Soluble High Throughput Assays

In one embodiment the invention provides soluble assays using moleculescorresponding to GPCR protein domains, such as ligand binding domain, anextracellular domain, a transmembrane domain (e.g., one comprising seventransmembrane regions and cytosolic loops), and a cytoplasmic domain, anactive site, a subunit association region, etc. Such domains may becovalently linked to a heterologous protein to create a chimericmolecule. In another embodiment, the invention provides solid phasebased in vitro assays in a high throughput format, where the domain,chimeric molecule, GPCR, or cell or tissue expressing an GPCR isattached to a solid phase substrate.

In certain high throughput assays, it is possible to screen up toseveral thousand different modulators or ligands in a single day. Inparticular, each well of a microtiter plate can be used to run aseparate assay against a selected potential modulator, or, ifconcentration or incubation time effects are to be observed, every 5-10wells can test a single modulator. Thus, a single standard microtiterplate can assay about 100 (e.g., 96) modulators.

The molecule of interest can be bound to the solid state component,directly or indirectly, via covalent or non covalent linkage e.g., via atag. The tag can be any of a variety of components. In general, amolecule which binds the tag (a tag binder) is fixed to a solid support,and the tagged molecule of interest (e.g., the signal transductionmolecule of interest) is attached to the solid support by interaction ofthe tag and the tag binder.

A number of tags and tag binders can be used, based upon known molecularinteractions well described in the literature. For example, where a taghas a natural binder, for example, biotin, protein A, or protein G, itcan be used in conjunction with appropriate tag binders (avidin,streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.)

Antibodies to molecules with natural binders such as biotin are alsowidely available and appropriate tag binders; see, SIGMA Immunochemicals1998 catalogue SIGMA, St. Louis Mo.).

Similarly, any haptenic or antigenic compound can be used in combinationwith an appropriate antibody to form a tag/tag binder pair. Thousands ofantibodies are commercially available and described in the literature.For example, in one common configuration, the tag is a first antibodyand the tag binder is a second antibody which recognizes the firstantibody. In addition to antibody-antigen interactions, receptor-ligandinteractions are also appropriate as tag and tag-binder pairs. Forexample, agonists and antagonists of cell membrane receptors (e.g., cellreceptor-ligand interactions such as transferrin, c-kit, viral receptorligands, cytokine receptors, chemokine receptors, interleukin receptors,immunoglobulin receptors and antibodies, the cadherein family, theintegrin family, the selectin family, and the like; see, e.g., Pigott &Power, The Adhesion Molecule Facts Book I (1993). Similarly, toxins andvenoms, viral epitopes, hormones (e.g., opiates, steroids, etc.),intracellular receptors (e.g. which mediate the effects of various smallligands, including steroids, thyroid hormone, retinoids and vitamin D;peptides), drugs, lectins, sugars, nucleic acids (both linear and cyclicpolymer configurations), oligosaccharides, proteins, phospholipids andantibodies can all interact with various cell receptors.

Synthetic polymers, such as polyurethanes, polyesters, polycarbonates,polyureas, polyamides, polyethyleneimines, polyarylene sulfides,polysiloxanes, polyimides, and polyacetates can also form an appropriatetag or tag binder. Many other tag/tag binder pairs are also useful inassay systems described herein, as would be apparent to one of skillupon review of this disclosure.

Common linkers such as peptides, polyethers, and the like can also serveas tags, and include polypeptide sequences of between about 5 and 200amino acids. Such flexible linkers are known to persons of skill in theart. For example, poly(ethelyne glycol) linkers are available fromShearwater Polymers, Inc. Huntsville, Ala. These linkers optionally haveamide linkages, sulfhydryl linkages, or heterofunctional linkages.

Tag binders are fixed to solid substrates using any of a variety ofmethods currently available. Solid substrates are commonly derivatizedor functionalized by exposing all or a portion of the substrate to achemical reagent which fixes a chemical group to the surface which isreactive with a portion of the tag binder. For example, groups which aresuitable for attachment to a longer chain portion would include amines,hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes andhydroxyalkylsilanes can be used to functionalize a variety of surfaces,such as glass surfaces. The construction of such solid phase biopolymerarrays is well described in the literature. See, e.g., Merrifield, J.Am. Chem. Soc. 85:2149-2154 (1963) (describing solid phase synthesis of,e.g., peptides); Geysen et al., J. Immun. Meth. 102:259-274 (1987)(describing synthesis of solid phase components on pins); Frank &Doring, Tetrahedron 44:60316040 (1988) (describing synthesis of variouspeptide sequences on cellulose disks); Fodor et al., Science,251:767-777 (1991); Sheldon et al., Clinical Chemistry 39(4):718-719(1993); and Kozal et al., Nature Medicine 2(7):753759 (1996) (alldescribing arrays of biopolymers fixed to solid substrates).Non-chemical approaches for fixing tag binders to substrates includeother common methods, such as heat, cross-linking by UV radiation, andthe like.

Treatment of Disease

In one aspect, the invention provides a method of treating a conditionassociated with a GPCR protein. Such a method involves administering toa subject in need of such treatment an effective amount of an antibodythat specifically binds to a structural feature of a GPCR protein. In apreferred embodiment of the invention, the antibody is raised against acell line expressing at least 150,000 copies of a GPCR protein per cell.

In one embodiment of the invention, the treatment involves administeringan effective amount of an antibody that is conjugated to a therapeuticentity. In a preferred embodiment of the invention, the antibody isconjugated to an anti-cancer therapeutic entity.

In one embodiment of the invention, the condition requiring treatmentthat is associated with a GPCR protein is neoplastic growth.

In another embodiment of the invention, the condition requiringtreatment that is associated with a GPCR protein is breast cancer.

In still another embodiment, an element or symptom of the conditionrequiring treatment that is associated with a GPCR protein isinflammation.

Drug Discovery

In one aspect, the invention provides methods for targeted drugdiscovery and pharmaceutical design based on secondary and tertiarystructures of GPCR proteins. In one embodiment of the invention,structural information on a GPCR protein is obtained from a study ofproteins isolated from cells expressing at least 150,000 copies of theGPCR protein per cell.

Modulators of GPCR activity may be tested using GPCR polypeptides. Thepolypeptide can be isolated, expressed in a cell, expressed in amembrane derived from a cell, expressed in tissue or in an animal,either recombinant or naturally occurring. For example, breast cancercells, normal prostate epithelial cells, placenta, testis tissue,transformed cells, or membranes can be used. Signal transduction canalso be examined in vitro with soluble or solid state reactions, or byusing a chimeric molecule such as an extracellular domain of a receptorcovalently linked to a heterologous signal transduction domain, or witha heterologous extracellular domain covalently linked to thetransmembrane and or cytoplasmic domain of a receptor. Geneamplification can also be examined. Furthermore, ligand-binding domainsof the protein of interest can be used in vitro in soluble or solidstate reactions to assay for ligand binding.

Test compounds can be tested for the ability to increase or decreaseGPCR activity of a GPCR polypeptide. The GPCR activity can be measuredafter contacting either a purified GPCR, a cell membrane preparation, oran intact cell with a test compound. A test compound which decreasesGPCR activity by at least about 10, preferably about 50, more preferablyabout 75, 90, or 100% is identified as a potential agent for decreasingGPCR activity. A test compound which increases GPCR activity by at leastabout 10, preferably about 50, more preferably about 75, 90, or 100% isidentified as a potential agent for increasing GPCR activity.

GPCR-Directed Compound Libraries

In one preferred embodiment, high throughput screening methods involveproviding a combinatorial chemical or peptide library containing a largenumber of potential therapeutic compounds (potential modulator or ligandcompounds). Such “combinatorial chemical libraries” or “ligandlibraries” are then screened in one or more assays, as described herein,to identify those library members (particular chemical species orsubclasses) that display a desired characteristic activity. Thecompounds thus identified can serve as conventional “lead compounds” orcan themselves be used as potential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis. For example, a linear combinatorial chemical library such asa polypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

Preparation and screening of combinatorial chemical and biochemicallibraries is well known to those of skill in the art. Such combinatorialchemical libraries include, but are not limited to, peptide libraries(see, e.g., U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res.37:487-493 (1991) and Houghton et al., Nature 354:84-88 (1991)). Otherchemistries for generating chemical diversity libraries can also beused. Such chemistries include, but are not limited to: peptoids (e.g.,PCT Publication NO: WO 91/19735), encoded peptides (e.g., PCTPublication WO 93/20242), random bio-oligomers (e.g., PCT PublicationNO: WO 92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514),diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs etal., Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogouspolypeptides (Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)),nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al.,J. Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic synthesesof small compound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661(1994)), oligocarbamates (Cho et al., Science 261:1303 (1993)), and/orpeptidyl phosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)),nucleic acid libraries (see Ausubel, Berger and Sambrook, all supra),peptide nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083),antibody libraries (see, e.g., Vaughn et al., Nature Biotechnology,14(3):309-314 (1996) and PCT/US96/10287), carbohydrate libraries (see,e.g., Liang et al., Science, 274:1520-1522 (1996) and U.S. Pat. No.5,593,853), small organic molecule libraries (see, e.g.,benzodiazepines, Baum C&EN, January 18, page 33 (1993); isoprenoids,U.S. Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat.No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134;morpholino compounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S.Pat. No. 5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem. Tech, LouisvilleKy., Symphony, Rainin, Wobum, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition,numerous combinatorial libraries are themselves commercially available(see, e.g., ComGenex, Princeton, N.J., Tripos, Inc., St. Louis, Mo., 3DPharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).

Lead-finding libraries can follow molecular mimicry principles and usethe substantial medicinal chemistry knowledge that has been generatedduring the last decade around GPCR compounds. Also useful in drug designare lead/drug likeness and computational combinatorial library design.

Studies suggest that certain classes and structural properties ofligands are important in designing drugs for particular receptors. Forexample, divalent ligands often selectively target opioid receptorheterodimers, while protein mimetics with β-turn/α-helix domains areimportant features for drugs mimicking hormones such as somatostatin andangiotensin. Cyclic α-peptides and β/γ peptides may also serve as abasis for drug design for some GPCRs.

The use of privileged substructures or molecular master keys that aretarget-class-specific or that mimic protein secondary structure elementsis a commonly used strategy in the art. The privileged-structureapproach utilizes molecular scaffolds or selected substructures able toprovide high-affinity ligands for diverse receptor targets. Empiricallyderived privileged structures include spiropiperidines,biphenyl-tetrazoles, benzimidazoles, and benzofurans. Chemoinformaticsmethods may also enable the automatic identification and extraction ofprivileged structures, which is particularly important for developingknowledge from high throughput screening data. Software such as ScitegicPipeline Pilot can also be used to generate a data-pipelining protocolthat generates frequency analysis based on the input of differentreference sets.

Thematic analysis may also be used to develop drugs for GPCR proteins.In this method, a class of proteins, such as a GPCR family, is analyzedto develop a classification based on the pairing of “sequence themes”and ligand structural motifs. A sequence theme is a consensus collectionof amino acids within the central binding cavity, and a motif is aspecific structural element binding to such a particularmicroenvironment of the binding site. This compilation of themes andmotifs can then be used to generate focused discovery libraries and toincrease the lead optimization efficiency for the drug targets.

Individual compound libraries that target subsets of GPCRs, such asorphan receptors, share a predefined combination of themes consisting ofa central dominant theme and peripheral ancillary themes. The libraryscaffold can then be designed to complement the central theme, withincorporation of a variety of structural motifs that address theindividual sequence themes. Such libraries, consisting of approximately1000 compounds, can then be thought of as representing a number ofpredefined themes which are either present or absent in a givenreceptor. This “fingerprinting” approach allows a score to be assignedto a particular library of compounds as to appropriateness of that groupof compounds for a particular receptor. Thematic analysis could also beused to develop new combinations of used and unused themes to increaseaffinity and selectivity of lead compounds in a pipeline.

Other design strategies include related computer-assisted drug design,which makes use of selected reference compound sets and moleculardescriptors together with cheminformatics methods to compare and rankthe similarity of designed candidate molecules. Homology-basedsimilarity searching can also identify potential ligands for orphanreceptors. Artificial neural networks, self-organizing maps, and supportvector machines may also be used in the drug design process—thesemethods align chemical and biological spaces based on mapping proceduresto determine which parts of the chemical-property space correspond tospecific target-families or therapeutic activities.

Administration and Pharmaceutical Compositions

GPCR modulators can be administered directly to the mammalian subjectfor modulation of signal transduction in vivo, e.g., for the treatmentof a cancer such as breast cancer. Administration is by any of theroutes normally used for introducing a modulator compound to the tissueto be treated. GPCR modulators can be administered in any suitablemanner, optionally with pharmaceutically acceptable carriers. Suitablemethods of administering such modulators are available and well known tothose of skill in the art, and, although more than one route can be usedto administer a particular composition, a particular route can oftenprovide a more immediate and more effective reaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention (see, e.g., Remington's Pharmaceutical Sciences,17.sup.th ed. 1985)).

GPCR modulators, alone or in combination with other suitable components,can be made into aerosol formulations (i.e., they can be “nebulized”) tobe administered via inhalation. Aerosol formulations can be placed intopressurized acceptable propellants, such as dichlorodifluoromethane,propane, nitrogen, and the like.

Formulations suitable for administration include aqueous and non-aqueoussolutions, isotonic sterile solutions, which can contain antioxidants,buffers, bacteriostats, and solutes that render the formulationisotonic, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. In the practice of this invention, compositions canbe administered, for example, by orally, topically, intravenously,intraperitoneally, intravesically or intrathecally. Optionally, thecompositions are administered orally or nasally. The formulations ofcompounds can be presented in unit-dose or multi-dose sealed containers,such as ampules and vials. Solutions and suspensions can be preparedfrom sterile powders, granules, and tablets of the kind previouslydescribed. The modulators can also be administered as part a of preparedfood or drug.

The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial response in thesubject over time. Such doses are administered prophylactically or to anindividual already suffering from the disease. The compositions areadministered to a patient in an amount sufficient to elicit an effectiveprotective or therapeutic response in the patient. An amount adequate toaccomplish this is defined as “therapeutically effective dose.” The dosewill be determined by the efficacy of the particular GPCR modulators(e.g., GPCR antagonists and anti-GPCR antibodies) employed and thecondition of the subject, as well as the body weight or surface area ofthe area to be treated. The size of the dose will also be determined bythe existence, nature, and extent of any adverse side-effects thataccompany the administration of a particular compound or vector in aparticular subject.

In determining the effective amount of the modulator to be administereda physician may evaluate circulating plasma levels of the modulator,modulator toxicities, and the production of anti-modulator antibodies.In general, the dose equivalent of a modulator is from about 1 ng/kg to10 mg/kg for a typical subject.

GPCR modulators of the present invention can be administered at a ratedetermined by the LD-50 of the modulator, and the side-effects of theinhibitor at various concentrations, as applied to the mass and overallhealth of the subject. Administration can be accomplished via single ordivided doses.

Purification of GPCRs from Recombinant Cells

Recombinant proteins are expressed by transformed bacteria or eukaryoticcells such as CHO cells or insect cells in large amounts, typicallyafter promoter induction, but expression can be constitutive. Promoterinduction with IPTG is a one example of an inducible promoter system.Cells are grown according to standard procedures in the art. Fresh orfrozen cells can be used for isolation of protein.

Proteins expressed in bacteria may form insoluble aggregates (“inclusionbodies”). Several protocols are suitable for purification of GPCRinclusion bodies. For example, purification of inclusion bodiestypically involves the extraction, separation and/or purification ofinclusion bodies by disruption of bacterial cells, e.g., by incubationin a buffer of 50 mM TRIS/HCL pH 7.5, 50 mM NaCl, 5 mM MgCl₂, 1 mM DTT,0.1 mM ATP, and 1 mM PMSF. The cell suspension can be lysed using 2-3passages through a French Press, homogenized using a Polytron (BrinkmanInstruments) or sonicated on ice. Alternative methods of lysing bacteriaare apparent to those of skill in the art (see, e.g., Sambrook et al.,supra; Ausubel et al., supra).

If necessary, inclusion bodies are solubilized, and the lysed cellsuspension is typically centrifuged to remove unwanted insoluble matter.Proteins that form inclusion bodies may be renatured by elution ordialysis with a compatible buffer. Suitable solvents include, but arenot limited to urea (from about 4 M to about 8 M), formamide (at leastabout 80%, volume/volume basis), and guanidine hydrochloride (from about4 M to about 8 M). Some solvents which are capable of solubilizingaggregate-forming proteins, for example SDS (sodium dodecyl sulfate),70% formic acid, are inappropriate for use in this procedure due to thepossibility of irreversible denaturation of the proteins, accompanied bya lack of immunogenicity and/or activity. Although guanidinehydrochloride and similar agents are denaturants, this denaturation isnot irreversible and renaturation may occur upon removal (by dialysis,for example) or dilution of the denaturant, allowing re-formation ofimmunologically and/or biologically active protein. Other suitablebuffers are known to those skilled in the art. The GPCR is separatedfrom other bacterial proteins by standard separation techniques, e.g.,with Ni-NTA agarose resin.

It is also possible to purify the GPCR from bacteria periplasm. Afterlysis of the bacteria, when the GPCR is exported into the periplasm ofthe bacteria, the periplasmic fraction of the bacteria can be isolatedby cold osmotic shock, as well as by other methods known to those ofskill in the art. To isolate recombinant proteins from the periplasm,the bacterial cells are centrifuged to form a pellet. The pellet isresuspended in a buffer containing 20% sucrose. To lyse the cells, thebacteria are centrifuged and the resultant pellet is resuspended inice-cold 5 mM MgSO.sub.4 and kept in an ice bath for approximately 10minutes. The cell suspension is centrifuged and the supernatant decantedand saved. The recombinant proteins present in the supernatant can beseparated from the host proteins by standard separation techniques wellknown to those of skill in the art.

Solubility Fractionation

Often as an initial step, particularly if the protein mixture iscomplex, an initial salt fractionation can separate many of the unwantedhost cell proteins (or proteins derived from the cell culture media)from the recombinant protein of interest. The preferred salt is ammoniumsulfate. Ammonium sulfate precipitates proteins by effectively reducingthe amount of water in the protein mixture. Proteins then precipitate onthe basis of their solubility. The more hydrophobic a protein is, themore likely it is to precipitate at lower ammonium sulfateconcentrations. A typical protocol includes adding saturated ammoniumsulfate to a protein solution so that the resultant ammonium sulfateconcentration is between 20-30%. This concentration will precipitate themost hydrophobic of proteins. The precipitate is then discarded (unlessthe protein of interest is hydrophobic) and ammonium sulfate is added tothe supernatant to a concentration known to precipitate the protein ofinterest. The precipitate is then solubilized in buffer and the excesssalt removed if necessary, either through dialysis or diafiltration.Other methods that rely on solubility of proteins, such as cold ethanolprecipitation, are well known to those of skill in the art and can beused to fractionate complex protein mixtures.

Size Differential Filtration

The molecular weight of GPCR can be used to isolated it from proteins ofgreater and lesser size using ultrafiltration through membranes ofdifferent pore size (for example, Amicon or Millipore membranes). As afirst step, the protein mixture is ultrafiltered through a membrane witha pore size that has a lower molecular weight cut-off than the molecularweight of the protein of interest. The retentate of the ultrafiltrationis then ultrafiltered against a membrane with a molecular cut offgreater than the molecular weight of the protein of interest. Therecombinant protein will pass through the membrane into the filtrate.The filtrate can then be chromatographed.

Column Chromatography

GPCRs can also be separated from other proteins on the basis of itssize, net surface charge, hydrophobicity, and affinity for ligands usingtechniques of column chromatography known in the art. In addition,antibodies raised against proteins can be conjugated to column matricesand the proteins immunopurified. All of these methods are well known inthe art. It will be apparent to one of skill that chromatographictechniques can be performed at any scale and using equipment from manydifferent manufacturers (e.g., Pharmacia Biotech).

Suitable test compounds for use in the screening assays of the inventioncan be obtained from any suitable source, e.g., conventional compoundlibraries. The test compounds can also be obtained using any of thenumerous approaches in combinatorial library methods known in the art,including: biological libraries; spatially addressable parallel solidphase or solution phase libraries; synthetic library methods requiringdeconvolution; the “one-bead one-compound” library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary approach is limited to peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds [Lam, (1997)]. Examples of methods forthe synthesis of molecular libraries can be found in the art. Librariesof compounds may be presented in solution or on beads, bacteria, spores,plasmids or phage.

Quantification of Protein Production

Western blot (immunoblot) analysis can be used to detect and quantifythe presence of -GPCR in the sample. The technique generally comprisesseparating sample proteins by gel electrophoresis on the basis ofmolecular weight, transferring the separated proteins to a suitablesolid support, (such as a nitrocellulose filter, a nylon filter, orderivitzed nylon filter), and incubating the sample with the antibodiesthat specifically bind GPCR. The anti-GPCR antibodies specifically bindto the GPCR on the solid support. These antibodies may be directlylabeled or alternatively may be subsequently detected using labeledantibodies (e.g., labeled sheep anti-mouse antibodies) that specificallybind to the anti-GPCR antibodies.

Labels

The particular label or detectable group used in an assay is not acritical aspect of the invention, as long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field and, in general, almost any label useful in assay methods canbe applied to the present invention. Thus, a label is any compositiondetectable by spectroscopic, photochemical, biochemical, immunochemical,electrical, optical or chemical means. Useful labels in the presentinvention include magnetic beads (e.g., DYNABEADS™), fluorescent dyes(e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like),radiolabels (e.g., ³H, ¹²⁵I, 35S, ¹⁴C, or ³²P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase and others commonly used in anELISA), and calorimetric labels such as colloidal gold or colored glassor plastic beads (e.g., polystyrene, polypropylene, latex, etc.).

The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. The choice oflabel used in an assay depends on factors such as the requiredsensitivity, ease of conjugation with the compound, stabilityrequirements, available instrumentation, and disposal provisions.

Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the molecule.The ligand then binds to another molecules (e.g., streptavidin)molecule, which is either inherently detectable or covalently bound to asignal system, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. The ligands and their targets can be used inany suitable combination with antibodies that recognize GPCRs, orsecondary antibodies that recognize anti-GPCR.

The molecules can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorophore. Enzymesof interest as labels will primarily be hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidotases, particularlyperoxidases. Fluorescent compounds include fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazined-iones, e.g., luminol. For a review of variouslabeling or signal producing systems that may be used, see U.S. Pat. No.4,391,904.

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple colorimetriclabels may be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

Some assay formats do not require the use of labeled components. Forinstance, agglutination assays can be used to detect the presence of thetarget antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

In addition, techniques developed for the production of “chimericantibodies”, the splicing of antibody genes from different species toobtain a molecule with appropriate antigen specificity and biologicalactivity can be used. Monoclonal and other antibodies can be “humanized”to prevent a patient from mounting an immune response against theantibody when it is used therapeutically. Such antibodies may besufficiently similar in sequence to human antibodies to be used directlyin therapy or may require alteration of a few key residues. For example,sequence differences between rodent antibodies and human sequences canbe minimized by replacing residues which differ from those in the humansequences by site directed mutagenesis of individual residues or bygrating of entire complementarity determining regions. Antibodies whichspecifically bind to GPCR can thus contain antigen binding sites whichare either partially or fully humanized, as disclosed in U.S. Pat. No.5,565,332.

Alternatively, techniques described for the production of single chainantibodies can be adapted using methods known in the art to producesingle chain antibodies which specifically bind to GPCR. Antibodies withrelated specificity, but of distinct idiotypic composition, can begenerated by chain shuffling from random combinatorial immunoglobinlibraries. Single-chain antibodies also can be constructed using a DNAamplification method, such as PCR, using hybridoma cDNA as a template.Single-chain antibodies can be mono- or bispecific, and can be bivalentor tetravalent. Construction of tetravalent, bispecific single-chainantibodies is taught. A nucleotide sequence encoding a single-chainantibody can be constructed using manual or automated nucleotidesynthesis, cloned into an expression construct using standardrecombinant DNA methods, and introduced into a cell to express thecoding sequence, as described below. Alternatively, single-chainantibodies can be produced directly using, for example, filamentousphage technology.

Antibodies according to the invention can be purified by methods wellknown in the art. For example, antibodies can be affinity purified bypassage over a column to which GPCR is bound. The bound antibodies canthen be eluted from the column using a buffer with a high saltconcentration

Immunoassays

In addition to the detection of GPCR genes and gene expression usingnucleic acid hybridization technology, one can also use immunoassays todetect GPCRs, e.g., to identify cells such as cancer cells, inparticular breast cancer cells, and variants of GPCRs. Immunoassays canbe used to qualitatively or quantitatively analyze GPCRs. A generaloverview of the applicable technology can be found in Harlow & Lane,Antibodies: A Laboratory Manual (1988).

Immunoassays use a labeling agent to specifically bind to and label thecomplex formed by the antibody and antigen. The labeling agent mayitself be one of the moieties comprising the antibody/antigen complex.Alternatively, the labeling agent may be a third moiety, such asecondary antibody that specifically binds to the antibody/GPCR complex(a secondary antibody is typically specific to antibodies of the speciesfrom which the first antibody is derived). Other proteins capable ofspecifically binding immunoglobulin constant regions, such as protein Aor protein G may also be used as the label agent. These proteins exhibita strong non-immunogenic reactivity with immunoglobulin constant regionsfrom a variety of species (see, e.g., Kronval et al., J. Immunol. 111:1401-1406 (1973); Akerstrom et al., J. Immunol. 135:2589-2542 (1985)).The labeling agent can be modified with a detectable moiety, such asbiotin, to which another molecule can specifically bind, such asstreptavidin. A variety of detectable moieties are well known to thoseskilled in the art.

Noncompetitive immunoassays are assays in which the amount of antigen isdirectly measured. In one preferred “sandwich” assay, for example, theanti-GPCR antibodies can be bound directly to a solid substrate on whichthey are immobilized. These immobilized antibodies then capture GPCRspresent in the test sample. The GPCR thus immobilized is then bound by alabeling agent, such as a second GPCR antibody bearing a label.Alternatively, the second antibody may lack a label, but it may, inturn, be bound by a labeled third antibody specific to antibodies of thespecies from which the second antibody is derived. The second or thirdantibody is typically modified with a detectable moiety, such as biotin,to which another molecule specifically binds, e.g., streptavidin, toprovide a detectable moiety.

In competitive assays, the amount of GPCR present in the sample ismeasured indirectly by measuring the amount of a known, added(exogenous) GPCR displaced (competed away) from an anti-GPCR antibody bythe unknown GPCR present in a sample. In one competitive assay, a knownamount of GPCR is added to a sample and the sample is then contactedwith an antibody that specifically binds to the GPCR. The amount ofexogenous GPCR bound to the antibody is inversely proportional to theconcentration of GPCR present in the sample. In a particularly preferredembodiment, the antibody is immobilized on a solid substrate. The amountof GPCR bound to the antibody may be determined either by measuring theamount of GPCR present in a GPCR/antibody complex, or alternatively bymeasuring the amount of remaining uncomplexed protein. The amount ofGPCR may be detected by providing a labeled GPCR molecule.

A hapten inhibition assay is another preferred competitive assay. Inthis assay the known GPCR is immobilized on a solid substrate. A knownamount of anti-GPCR antibody is added to the sample, and the sample isthen contacted with the immobilized GPCR. The amount of anti-GPCRantibody bound to the known immobilized GPCR is inversely proportionalto the amount of GPCR present in the sample. Again, the amount ofimmobilized antibody may be detected by detecting either the immobilizedfraction of antibody or the fraction of the antibody that remains insolution. Detection may be direct where the antibody is labeled orindirect by the subsequent addition of a labeled moiety thatspecifically binds to the antibody as described above.

Immunoassays in the competitive binding format can also be used forcrossreactivity determinations. For example, a protein at leastpartially encoded by SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, or 17 can beimmobilized to a solid support. Proteins (e.g., GPCR proteins andhomologs) are added to the assay that compete for binding of theantisera to the immobilized antigen. The ability of the added proteinsto compete for binding of the antisera to the immobilized protein iscompared to the ability of GPCRs encoded by SEQ ID NO: 1, 3, 5, 7, 9,11, 13, 15, or 17 to compete with itself. The percent crossreactivityfor the above proteins is calculated, using standard calculations. Thoseantisera with less than 10% crossreactivity with each of the addedproteins listed above are selected and pooled. The cross-reactingantibodies are optionally removed from the pooled antisera byimmunoabsorption with the added considered proteins, e.g., distantlyrelated homologs.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described above to compare a second protein,thought to be perhaps an allele or polymorphic variant of an GPCR, tothe immunogen protein (i.e., the GPCR of SEQ ID NOS: 1, 3, 5, 7, 9, 11,13, 15, or 17). In order to make this comparison, the two proteins areeach assayed at a wide range of concentrations and the amount of eachprotein required to inhibit 50% of the binding of the antisera to theimmobilized protein is determined. If the amount of the second proteinrequired to inhibit 50% of binding is less than 10 times the amount ofthe protein encoded by SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, or 17 thatis required to inhibit 50% of binding, then the second protein is saidto specifically bind to the polyclonal antibodies generated to a GPCRimmunogen.

One of skill in the art will appreciate that it is often desirable tominimize non-specific binding in immunoassays. Particularly, where theassay involves an antigen or antibody immobilized on a solid substrateit is desirable to minimize the amount of non-specific binding to thesubstrate. Means of reducing such non-specific binding are well known tothose of skill in the art. Typically, this technique involves coatingthe substrate with a proteinaceous composition. In particular, proteincompositions such as bovine serum albumin (BSA), nonfat powdered milk,and gelatin are widely used with powdered milk being most preferred.

Other assay formats include liposome immunoassays (LIA), which useliposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers upon a binding event. Thereleased chemicals are then detected according to standard techniques(see Monroe et al., Amer. Clin. Prod. Rev. 5:34-41 (1986)).

Deorphanization

Among known GPCR proteins some 200 exist for which their natural ligandis unknown. These “orphan” GPCRs have the potential to serve as targetsfor novel drugs and novel indications. Yet, to be used in pharmaceuticalresearch, these GPCRs need first to be “deorphanized”, i.e. theirnatural ligands need to be discovered to allow for development ofapplications such as high-throughput screening assays.

Deorphanization refers to the identification of activating ligands is akey task in reverse molecular pharmacology. Identifying receptor-agonistpairs usually allows the rapid elucidation of the physiological role ofboth partners, sometimes putting them in unexpected contexts. Althoughbioinformatics methods are initially helpful to successfully directligand-pairing experiments, deorphanization strategies generally rely onbiological screening of orphan GPCRs expressed in recombinant expressionsystems such as immortalized mammalian cells, yeast, and Xenopusmelanophores.

Agonist ligand libraries used for deorphanization can include smallmolecules, peptides, proteins, lipids or tissue extracts. Identificationof an activating agonistic ligand of the cell-surface-expressedreceptors is often dependent on the activation of an intracellularsignaling cascade.

A difficulty in assay design for orphan GPCRs is that the signalingcascade is not known for a new orphan receptor. Therefore, generic assaysystems amenable for high throughput screening are generally used toscreen large surrogate ligand collections. One successful approach fordeorphanization uses fluorescent imaging plate reader (FLIPR) screeningtechnology, which detects ligand-induced intracellular Ca⁺²mobilization.

Given the broad chemical diversity of the molecules that are recognizedby GPCRs, deorphanization libraries try to cover as many known activechemical classes as possible. The term “surrogate agonist library” isalso appropriate given that the purpose of these libraries is to find achemical compound that selectively activates a given orphan receptor ofinterest. Typically, compounds identical or similar to previouslyidentified GPCR agonists are included together with approved drugs andother reference compounds with known bioactivity, such as primarymetabolites like the KEGG compound set, or commercially availablecompilations like the Tocris LOPAC, the Prestwick, or the Sial Biomolsets.

Typically, the size of deorphanization or surrogate libraries is on theorder of a few thousand well-characterized compounds amenable formedium-throughput screening. The design of lead-finding librariesfollows the same molecular mimicry principles and makes best use of thesubstantial medicinal chemistry knowledge generated during the lastdecades around GPCR compounds together with more modern concepts,including lead/drug likeness and computational combinatorial librarydesign. Focused library design concepts target the classical bindingsites in general, while design concepts of bivalent ligands andallosteric ligands can be used as the understanding of the GPCRoligomerization phenomenon increases.

Diseases Associated with GPCRs

Mutations in GPCRs have been associated with a wide variety ofillnesses, particularly cancers and diseases involving inflammation.

Some disease-causing mutations result in constitutive receptoractivation, such as in Jansen's disease, where the hypercalcemia andskeletal dysplasia found in many cases is the result of a constitutivelyoveractive parathyroid hormone/parathyroid hormone related proteinreceptor. In such diseases, inhibitors of activation are of particularinterest as potential therapeutics.

Virally encoded GPCRs may also have a direct role in human diseases. Forexample, Kaposi's sarcoma-associated herpes virus has been implicated inKaposi's sarcomagenesis, and the human cytomegalovirus-encoded GPCRshave been implicated in atherosclerosis.

Certain GPCRs are associated with the disorders of the peripheral andcentral nervous system (CNS), cardiovascular diseases, hematologicaldiseases, cancer, inflammation, urological diseases, respiratorydiseases and gastroenterological diseases. Such disorders may include awide range of diseases, as discussed further below.

Nervous System Disorders

CNS disorders include disorders of the central nervous system as well asdisorders of the peripheral nervous system.

CNS disorders include, but are not limited to brain injuries,cerebrovascular diseases and their consequences, Parkinson's disease,corticobasal degeneration, motor neuron disease, dementia, includingALS, multiple sclerosis, traumatic brain injury, stroke, post-stroke,post-traumatic brain injury, and small-vessel cerebrovascular disease.Dementias, such as Alzheimer's disease, vascular dementia, dementia withLewy bodies, frontotemporal dementia and Parkinsonism linked tochromosome 17, frontotemporal dementias, including Pick's disease,progressive nuclear palsy, corticobasal degeneration, Huntington'sdisease, thalamic degeneration, Creutzfeld-Jakob dementia, HIV dementia,schizophrenia with dementia, and Korsakoff's psychosis, within themeaning of the definition are also considered to be CNS disorders. JakobSimilarly, cognitive-related disorders, such as mild cognitiveimpairment, age-associated memory impairment, age-related cognitivedecline, vascular cognitive impairment, attention deficit disorders,attention deficit hyperactivity disorders, and memory disturbances inchildren with learning disabilities are also considered to be CNSdisorders. Jakob Pain, within the meaning of this definition, is alsoconsidered to be a CNS disorder. Pain can be associated with CNSdisorders, such as multiple sclerosis, spinal cord injury, sciatica,failed back surgery syndrome, traumatic brain injury, epilepsy,Parkinson's disease, post-stroke, and vascular lesions in the brain andspinal cord (e.g., infarct, hemorrhage, vascular malformation).Non-central neuropathic pain includes that associated with postmastectomy pain, phantom feeling, reflex sympathetic dystrophy (RSD),trigeminal neuralgiaradioculopathy, post-surgical pain, HIV/AIDS relatedpain, cancer pain, metabolic neuropathies (e.g., diabetic neuropathy,vasculitic neuropathy secondary to connective tissue disease),paraneoplastic polyneuropathy associated, for example, with carcinoma oflung, or leukemia, or lymphoma, or carcinoma of prostate, colon orstomach, trigeminal neuralgia, cranial neulalgias, and post-herpeticneuralgia. Pain associated with peripheral nerve damage, central pain(i.e. due to cerebral ischemia) and various chronic pain i.e., lumbago,back pain (low back pain), inflammatory and/or rheumatic pain. Headachepain (for example, migraine with aura, migraine without aura, and othermigraine disorders), episodic and chronic tension-type headache,tension-type like headache, cluster headache, and chronic paroxysmalhemicrania are also CNS disorders. Jakob Visceral pain such aspancreatits, intestinal cystitis, dysmenorrhea, irritable Bowelsyndrome, Crohn's disease, biliary colic, ureteral colic, myocardialinfarction and pain syndromes of the pelvic cavity, e.g., vulvodynia,orchialgia, urethral syndrome and protatodynia are also CNS disorders.Jakob Also considered to be a disorder of the nervous system are acutepain, for example postoperative pain, and pain after trauma. Jakob Thehuman GPCR is highly expressed in the following brain tissues: brain,Alzheimer brain, cerebellum (right), cerebellum (left), cerebral cortex,Alzheimer brain frontal lobe, occipital lobe, pons, substantia nigra,cerebral meninges, corpus callosum, dorsal root ganglia, neuroblastomaIMR32 cells. The expression in brain tissues and in particular thedifferential expression between diseased tissue Alzheimer brain andhealthy tissue brain, between diseased tissue Alzheimer brain frontallobe and healthy tissue frontal lobe demonstrates that the human GPCR ormRNA can be utilized to diagnose nervous system diseases. Additionallythe activity of the human GPCR can be modulated to treat nervous systemdiseases.

Cardiovascular Disorders

GPCRs are highly expressed in the following cardiovascular relatedtissues: heart, pericardium, heart atrium (right), heart atrium (left),artery, coronary artery, coronary artery sclerotic. Expression in theabove mentioned tissues and in particular the differential expressionbetween diseased tissue coronary artery sclerotic and healthy tissuecoronary artery indicates that GPCR protein, DNA or mRNA can be utilizedto diagnose cardiovascular diseases.

Heart failure is defined as a pathophysiological state in which anabnormality of cardiac function is responsible for the failure of theheart to pump blood at a rate commensurate with the requirement of themetabolizing tissue. It includes all forms of pumping failures such ashigh-output and low-output, acute and chronic, right-sided orleft-sided, systolic or diastolic, independent of the underlying cause.

Myocardial infarction (MI) is generally caused by an abrupt decrease incoronary blood flow that follows a thrombotic occlusion of a coronaryartery previously narrowed by arteriosclerosis. MI prophylaxis (primaryand secondary prevention) is included as well as the acute treatment ofMI and the prevention of complications. Ischemic diseases are conditionsin which the coronary flow is restricted resulting in a perfusion whichis inadequate to meet the myocardial requirement for oxygen. This groupof diseases includes stable angina, unstable angina and asymptomaticischemia.

Arrhythmias include all forms of atrial and ventricular tachyarrhythmas,atrial tachycardia, atrial flutter, atrial fibrillation,atrio-ventricular reentrant tachycardia, preexitation syndrome,ventricular tachycardia, ventricular flutter, ventricular fibrillation,as well as bradycardic forms of arrhythmias.

Hypertensive vascular diseases include primary as well as all kinds ofsecondary arterial hypertension, renal, endocrine, neurogenic, others.The genes may be used as drug targets for the treatment of hypertensionas well as for the prevention of all complications arising fromcardiovascular diseases.

Peripheral vascular diseases are defined as vascular diseases in whicharterial and/or venous flow is reduced resulting in an imbalance betweenblood supply and tissue oxygen demand. It includes chronic peripheralarterial occlusive disease (PAOD), acute arterial thrombosis andembolism, inflammatory vascular disorders, Raynaud's phenomenon andvenous disorders.

Cardiovascular diseases include but are not limited to disorders of theheart and the vascular system like congestive heart failure, myocardialinfarction, ischemic diseases of the heart, all kinds of atrial andventricular arrhythmias, hypertensive vascular diseases, peripheralvascular diseases, and atherosclerosis.

Examples of disorders of lipid metabolism are hyperlipidemia (abnormallyhigh levels of fats (cholesterol, triglycerides, or both) in the blood,may be caused by family history of hyperlipidemia), obesity, a high-fatdiet, lack of exercise, moderate to high alcohol consumption, cigarettesmoking, poorly controlled diabetes, and an underactive thyroid gland),hereditary hyperlipidemias (type I hyperlipoproteinemia (familialhyperchylomicronemia), type II hyperlipoproteinemia (familialhypercholesterolemia), type In hyperlipoproteinemia, type IVhyperlipoproteinemia, or type V hyperlipoproteinemia),hypolipoproteinemia, lipidoses (caused by abnormalities in the enzymesthat metabolize fats), Gaucher's disease, Niemann-Pick disease, Fabry'sdisease, Wolman's disease, cerebrotendinous xanthomatosis,sitosterolemia, Refsum's disease, or Tay-Sachs disease.

Kidney Disorders

Kidney disorders may lead to hypertension or hypotension. Examples ofkidney problems possibly leading to hypertension are renal arterystenosis, pyelonephritis, glomerulonephritis, kidney tumors, polycistickidney disease, injury to the kidney, or radiation therapy affecting thekidney. Excessive urination may lead to hypotension.

Hematological Disorders

Hematological disorders comprise diseases of the blood and all itsconstituents as well as diseases of organs and tissues involved in thegeneration or degradation of all the constituents of the blood. Theyinclude but are not limited to 1) Anemias, 2) MyeloproliferativeDisorders, 3) Hemorrhagic Disorders, 4) Leukopenia, 5) EosinophilicDisorders, 6) Leukemias, 7) Lymphomas, 8) Plasma Cell Dyscrasias, 9)Disorders of the Spleen in the course of hematological disorders.Disorders according to 1) include, but are not limited to anemias due todefective or deficient hem synthesis, deficient erytlhropoiesis.Disorders according to 2) include, but are not limited to polycythemiavera, tumor-associated erythrocytosis, myelofibrosis, thrombocythemia.Disorders according to 3) include, but are not limited to vasculitis,thrombocytopenia, heparin-induced thrombocytopenia, thromboticthrombocytopenic purpura, hemolytic-uremic syndrome, hereditary andacquired disorders of platelet function, hereditary coagulationdisorders. Disorders according to 4) include, but are not limited toneutropenia, lymphocytopenia. Disorders according to 5) include, but arenot limited to hypereosinophilia, idiopathic hypereosinophilic syndrome.Disorders according to 6) include, but are not limited to acute myeloicleukemia, acute lymphoblastic leukemia, chronic myelocytic leukemia,chronic lymphocytic leukemia, myelodysplastic syndrome. Disordersaccording to 7) include, but are not limited to Hodgkin's disease,non-Hodgkin's lymphoma, Burkitt's lymphoma, mycosis fungoides cutaneousT-cell lymphoma. Disorders according to 8) include, but are not limitedto multiple myeloma, macroglobulinemia, heavy chain diseases. Inextension of the preceding idiopathic thrombocytopenic purpura, irondeficiency anemia, megaloblastic anemia (vitamin B12 deficiency),aplastic anemia, thalassemia, malignant lymphoma bone marrow invasion,malignant lymphoma skin invasion, hemolytic uremic syndrome, giantplatelet disease are considered to be hematological diseases too.

The human GPCR is highly expressed in the following tissues of thehematological system: leukocytes peripheral blood), bone marrow,erythrocytes, lymphnode, thymus, thrombocytes, bone marrow CD34+ cells,bone marrow CD15+ cells, spleen, spleen liver cirrhosis.

Gastrointestinal and Liver Diseases

Gastrointestinal diseases comprise primary or secondary, acute orchronic diseases of the organs of the gastrointestinal tract which maybe acquired or inherited, benign or malignant or metaplastic, and whichmay affect the organs of the gastrointestinal tract or the body as awhole. They comprise but are not limited to 1) disorders of theesophagus like achalasia, vigoruos achalasia, dysphagia, cricopharyngealincoordination, pre-esophageal dysphagia, diffuse esophageal spasm,globus sensation, Barrett's metaplasia, gastroesophageal reflux, 2)disorders of the stomach and duodenum like functional dyspepsia,inflammation of the gastric mucosa, gastritis, stress gastritis, chronicerosive gastritis, atrophy of gastric glands, metaplasia of gastrictissues, gastric ulcers, duodenal ulcers, neoplasms of the stomach, 3)disorders of the pancreas like acute or chronic pancreatitis,insufficiency of the exocrinic or endocrinic tissues of the pancreaslike steatorrhea, diabetes, neoplasms of the exocrine or endocrinepancreas like 3.1) multiple endocrine neoplasia syndrome, ductaladenocarcinoma, cystadenocarcinoma, islet cell tumors, insulinoma,gastrinoma, carcinoid tumors, glucagonoma, Zollinger-Ellison syndrome,Vipoma syndrome, malabsorption syndrome, 4) disorders of the bowel likechronic inflammatory diseases of the bowel, Crohn's disease, ileus,diarrhea and constipation, colonic inertia, megacblon, malabsorptionsyndrome, ulcerative colitis, 4.1) functional bowel disorders likeirritable bowel syndrome, 4.2) neoplasms of the bowel like familialpolyposis, adenocarcinoma, primary malignant lymphoma, carcinoid tumors,Kaposi's sarcoma, polyps, cancer of the colon and rectum.

Liver diseases comprise primary or secondary, acute or chronic diseasesor injury of the liver which may be acquired or inherited, benign ormalignant, and which may affect the liver or the body as a whole. Theycomprise but are not limited to disorders of the bilirubin metabolism,jaundice, syndroms of Gilbert's, Crigler-Najjar, Dubin-Johnson andRotor; intrahepatic cholestasis, hepatomegaly, portal hypertension,ascites, Budd-Chiari syndrome, portal-systemic encephalopathy, fattyliver, steatosis, Reye's syndrome, liver diseases due to alcohol,alcoholic hepatitis or cirrhosis, fibrosis and cirrhosis, fibrosis andcirrhosis of the liver due to inborn errors of metabolism or exogenoussubstances, storage diseases, syndromes of Gaucher's, Zellweger's,Wilson's-disease, acute or chronic hepatitis, viral hepatitis and itsvariants, inflammatory conditions of the liver due to viruses, bacteria,fungi, protozoa, helminths; drug induced disorders of the liver, chronicliver diseases like primary sclerosing cholangitis,alpha.sub.1-antitrypsin-deficiency, primary biliary cirrhosis,postoperative liver disorders like postoperative intrahepaticcholestasis, hepatic granulomas, vascular liver disorders associatedwith systemic disease, benign or malignant neoplasms of the liver,disturbance of liver metabolism in the new-born or prematurely born.

GPCRs are highly expressed in the following tissues of thegastro-enterological system: esophagus, esophagus tumor, stomach tumor,rectum. The expression in the above mentioned tissues and in particularthe differential expression between diseased tissue esophagus tumor andhealthy tissue esophagus, between diseased tissue stomach tumor andhealthy tissue stomach demonstrates that GPCR DNA, mRNA and polypeptidescan be utilized to diagnose of gastroenterological disorders.Additionally the activity of the human GPCR can be modulated to treatgastroenterological disorders.

Cancer Disorders

Cancer disorders within the scope of this definition comprise anydisease of an organ or tissue in mammals characterized by poorlycontrolled or uncontrolled multiplication of normal or abnormal cells inthat tissue and its effect on the body as a whole. Cancer diseaseswithin the scope of the definition comprise benign neoplasms,dysplasias, hyperplasias as well as neoplasms showing metastatic growthor any other transformations like e.g. leukoplakias which often precedea breakout of cancer. Cells and tissues are cancerous when they growmore rapidly than normal cells, displacing or spreading into thesurrounding healthy tissue or any other tissues of the body described asmetastatic growth, assume abnormal shapes and sizes, show changes intheir nucleocytoplasmatic ratio, nuclear polychromasia, and finally maycease.

Cancerous cells and tissues may affect the body as a whole when causingparaneoplastic syndromes or if cancer occurs within a vital organ ortissue, normal function will be impaired or halted, with possible fatalresults. The ultimate involvement of a vital organ by cancer, eitherprimary or metastatic, may lead to the death of the mammal affected.Cancer tends to spread, and the extent of its spread is usually relatedto an individual's chances of surviving the disease.

Cancers are generally said to be in one of three stages of growth:early, or localized, when a tumor is still confined to the tissue oforigin, or primary site; direct extension, where cancer cells from thetumor have invaded adjacent tissue or have spread only to regional lymphnodes; or metastasis, in which cancer cells have migrated to distantparts of the body from the primary site, via the blood or lymph systems,and have established secondary sites of infection.

Cancer is said to be malignant because of its tendency to cause death ifnot treated. Benign tumors usually do not cause death, although they mayif they interfere with a normal body function by virtue of theirlocation, size, or paraneoplastic side effects. Hence benign tumors fallunder the definition of cancer within the scope of this definition aswell. In general, cancer cells divide at a higher rate than do normalcells, but the distinction between the growth of cancerous and normaltissues is not so much the rapidity of cell division in the former as itis the partial or complete loss of growth restraint in cancer cells andtheir failure to differentiate into a useful, limited tissue of the typethat characterizes the functional equilibrium of growth of normaltissue.

The term “cancer” as referred to herein is not limited to simple benignneoplasia but comprises any other benign and malign neoplasia like 1)Carcinoma, 2) Sarcoma, 3) Carcinosarcoma, 4) Cancers of theblood-forming tissues, 5) tumors of nerve tissues including the brain,6) cancer of skin cells.

GPCRs are expressed in the following cancer tissues: esophagus tumor,stomach tumor, lung tumor, ovary tumor, kidney tumor. The expression inthe above mentioned tissues and in particular the differentialexpression between diseased tissue esophagus tumor and healthy tissueesophagus, between diseased tissue stomach tumor and healthy tissuestomach, between diseased tissue lung tumor and healthy tissue lung,between diseased tissue ovary tumor and healthy tissue ovary, betweendiseased tissue kidney tumor and healthy tissue kidney demonstrates thatthe human GPCR DNA, mRNA and polypeptides can be utilized to diagnose ofcancer. Additionally the activity of GPCR can be modulated to treatcancer.

Inflammatory Diseases

Inflammatory diseases comprise diseases triggered by cellular ornon-cellular mediators of the immune system or tissues causing theinflammation of body tissues and subsequently producing an acute orchronic inflammatory condition. Examples for such inflammatory diseasesare hypersensitivity reactions of type I-IV, for example but not limitedto hypersensitivity diseases of the lung including asthma, atopicdiseases, allergic rhinitis or conjunctivitis, angioedema of the lids,hereditary angioedema, antireceptor hypersensitivity reactions andautoimmune diseases, Hashimoto's thyroiditis, systemic lupuserythematosus, Goodpasture's syndrome, pemphigus, myasthenia gravis,Grave's and Raynaud's disease, type B insulin-resistant diabetes,rheumatoid arthritis, psoriasis, Crohn's disease, scleroderma, mixedconnective tissue disease, polymyositis, sarcoidosis,glomerulonephritis, acute or chronic host versus graft reactions.

GPCR is expressed in the following tissues of the immune system andtissues responsive to components of the immune system as well as in thefollowing tissues responsive to mediators of inflammation: leukocytes(peripheral blood), bone marrow, bone marrow CD15+ cells, spleen livercirrhosis, lung COPD. The expression in the above mentioned tissues andin particular the differential expression between diseased tissue spleenliver cirrhosis and healthy tissue spleen, between diseased tissue lungCOPD and healthy tissue lung demonstrates that GPCR DNA, mRNA andpolypeptides can be utilized to diagnose of inflammatory diseases.Additionally the activity of GPCR can be modulated to treat inflammatorydiseases.

Disorders Related to Pulmology

Asthma is thought to arise as a result of interactions between multiplegenetic and environmental factors and is characterized by three majorfeatures: 1) intermittent and reversible airway obstruction caused bybronchoconstriction, increased mucus production, and thickening of thewalls of the airways that leads to a narrowing of the airways, 2) airwayhyperresponsiveness, and 3) airway inflammation. Certain cells arecritical to the inflammatory reaction of asthma and they include T cellsand antigen presenting cells, B cells that produce IgE, and mast cells,basophils, eosinophils, and other cells that bind IgE. These effectorcells accumulate at the site of allergic reaction in the airways andrelease toxic products that contribute to the acute pathology andeventually to tissue destruction related to the disorder. Other residentcells, such as smooth muscle cells, lung epithelial cells,mucus-producing cells, and nerve cells may also be abnormal inindividuals with asthma and may contribute to its pathology. While theairway obstruction of asthma, presenting clinically as an intermittentwheeze and shortness of breath, is generally the most pressing symptomof the disease requiring immediate treatment, the inflammation andtissue destruction associated with the disease can lead to irreversiblechanges that eventually make asthma a chronic and disabling disorderrequiring long-term management.

Chronic obstructive pulnonary (or airways) disease (COPD) is a conditiondefined physiologically as airflow obstruction that generally resultsfrom a mixture of emphysema and peripheral airway obstruction due tochronic bronchitis [Botstein, 1980]. Emphysema is characterized bydestruction of alveolar walls leading to abnormal enlargement of the airspaces of the lung. Chronic bronchitis is defined clinically as thepresence of chronic productive cough for three months in each of twosuccessive years. In COPD, airflow obstruction is usually progressiveand is only partially reversible. By far the most important risk factorfor development of COPD is cigarette smoking, although the disease doesalso occur in non-smokers.

GPCR is highly expressed in the following tissues of the respiratorysystem: leukocytes (peripheral blood), bone marrow CD15+ cells, lung,lung right upper lobe, lung right mid lobe, lung right lower lobe, lungtumor, lung COPD, trachea. The expression in the above mentioned tissuesand in particular the differential expression between diseased tissuelung tumor and healthy tissue lung, between diseased tissue lung COPDand healthy tissue lung demonstrates that GPCR DNA, mRNA andpolypeptides can be utilized to diagnose of respiratory diseases.Additionally the activity of GPCR can be modulated to treat thosediseases.

Disorders Related to Urology

Genitourinary disorders comprise benign and malign disorders of theorgans constituting the genitourinary system of female and male, renaldiseases like acute or chronic renal failure, immunologically mediatedrenal diseases like renal transplant rejection, lupus nephritis, immunecomplex renal diseases, glomerulopathies, nephritis, toxic nephropathy,obstructive uropathies like benign prostatic hyperplasia (BPH),neurogenic bladder syndrome, urinary incontinence like urge-, stress-,or overflow incontinence, pelvic pain, and erectile dysfunction.

GPCR is expressed in the following urological tissues: ureter, penis,corpus cavernosum, fetal kidney, kidney, kidney tumor. The expression inthe above mentioned tissues and in particular the differentialexpression between diseased tissue kidney tumor and healthy tissuekidney demonstrates that GPCR DNA, mRNA and polypeptides can be utilizedto diagnose of urological disorders. Additionally the activity of thehuman GPCR can be modulated to treat urological disorders.

The present invention provides for both prophylacetic and therapeuticmethods for disorders of the peripheral and central nervous system,cardiovascular diseases, hematological diseases, cancer, inflammation,urological diseases, respiratory diseases and gastroenterologicaldiseases.

The present invention provides methods of treating an individualafflicted with a disease or disorder characterized by unwantedexpression or activity of GPCR or a protein in the GPCR signalingpathway. In one embodiment, the method involves administering an agentlike any agent identified or identifiable in assays as described herein,or a combination of such agents to modulate expression or activity ofGPCR or proteins in the GPCR signaling pathway. In another embodiment,the method involves administering a regulator of GPCR as therapy tocompensate for reduced or undesirably low expression or activity of GPCRor a protein in the GPCR signaling pathway.

The expression in the above mentioned tissues and in particular thedifferential expression between diseased tissue spleen liver cirrhosisand healthy tissue spleen demonstrates that GPCR DNA, mRNA andpolypeptides can be utilized to diagnose of hematological diseases.Additionally the activity of GPCR can be modulated to treathematological disorders.

EXAMPLES

The following examples are provided to illustrate the invention and arenot intended to limit its scope. Other variants of the invention will bereadily apparent to one of ordinary skill in the art and are encompassedby the appended claims.

Example I Expression Vector Design

A vector (pMEX2) was designed to facilitate expression and detection ofa GPCR expressed with correct orientation on the cytoplasmic membrane ofmammalian cells. The vector contained a pUC origin and thebeta-lactamase gene for replication and ampicillin selection of theplasmid in bacteria, as shown in FIG. 10. A puromycin resistance markerfor maintaining the plasmid in mammalian cells is also included in thevector. Expression of the gene of interest was under control of a strongCMV promoter for high-level transcription activity. The expressioncassette also contained a Kozak consensus sequence for optimaltranslation initiation and a SV40 late poly adenylation signal forstability of the transcripts.

Some features were added to the vector to facilitate isolation of GPCRcell lines. First, transportation of the receptor protein to thecytoplasmic membrane was greatly improved by fusion of an amino-terminalcleavable secretory signal peptide (amino acid seq:METDTLLLWVLLLWVPGSTGD, corresponding to SEQ ID NO: 19, position1102-1164) derived from a murine Ig kappa light chain. Second, a shortaffinity tag (Flag tag; amino acid seq: DYKDDDDK corresponding to SEQ IDNO: 19, position 1168-1191) was fused downstream from the signal peptideand followed by a short flexible linker (glycine-serine-glycine)upstream from the mature sequence of the target gene.

Addition of the Flag tag at the amino-terminus of the receptor servedtwo functions. First, it facilitated detection of the recombinantreceptor and isolation of cells expressing the receptor. Secondly, itserved as a marker for correct orientation of the receptor on thecytoplasmic membrane. Therefore, only receptors of correct orientationwith the Flag tag exposed to the extracellular side of the cytoplasmicmembrane can be detected by the mAb. The use of a universal affinity tagin this application is not limited to the Flag tag and can be extendedto any sequence (e.g. Flag, myc, H is tag, C9, HA etc.) which isrecognized by a fluorescence-labeled binder, as long as it does notinterfere with ligand binding to the fused receptor.

Upon translocation of the full-length protein to the membrane, thesignal peptide was processed, leaving the receptor with anamino-terminal Flag tag exposed to the extracellular environment. Openreading frame of the GPCR to be expressed was amplified by polymerasechain reaction (PCR) from either a cDNA library or an EST clone andsubcloned into the cloning sites (5′-BamH I and Sal I-3′) on the vector.The integrity of the gene sequence was confirmed by sequencing reactionsthrough the whole insert using primers outside the cloning sites on thevector. A typical vector map of pMEX2 and its nucleotide sequence areshown in FIG. 10 and FIG. 11 respectively.

Example II Screening for GPCR Clones

FACS analysis was carried out to confirm surface expression of theexogenously expressed receptors using the anti-Flag M2 mAb (Sigma) in atransient expression experiment. The plasmid DNA can be delivered intoany mammalian cells using any method, including, but not limited to,electroporation, lipid cation and calcium phosphate-mediatedtransfection, or retrovirus-mediated infection. For the currentexperiments, the vector was transfected into either human embryonickidney cells (HEK293) and its derivative (293T) or Chinese hamster ovarycells (CHO-K1) by Lipofectamine 2000 (Invitrogen) according to themanufacturer's protocol. Expression of the recombinant GPCR on cellsurface was detected 48 to 60 hours post transfection byfluorescence-activated cell sorting (FACS). A typical expression profilefor three GPCRs (MRGF, P2RY8 and GPR84) under transient expressioncondition is shown in FIG. 15 which shows broad spectrum of expressioncrossing two to three quadrants in the histograms.

Example III Ligand-Binding Activity of the Recombinant Receptors

A saturation ligand-binding assay was performed to confirm that therecombinant receptors retain its native conformation and ligand-bindingactivity. The average ligand-binding sites (Bmax; pmol/mg protein) andthe affinity (Kd; nM) of the exogenously expressed receptor in atransient expression experiment was measured at 12 radioligandconcentrations with duplicate total and non-specific bindingdeterminations. Representative result of specific binding for histaminereceptor H2 is shown in FIG. 16. The human receptor exhibited similaraffinity for the pig ligand as the endogenous receptor (data not shown).Based on the Bmax value and membrane protein expression per cell, theligand binding sites was calculated to be 1.05 million copies/cell.

Example IV Isolation of Stable Cell Lines Expressing High-Level GPCRS

Cell culture. HEK293 cells were maintained in DMEM supplemented with 10%FBS (Invitrogen Inc.), 100 U/ml penicillin/streptomycin. CHOK1 cellswere maintained in either DMEM/Ham's F-12 Mix or Ham's F-12 mediasupplemented with 10% FBS 100 U/ml penicillin/streptomycin (InvitrogenInc.). Cell transfections were carried out with Lipofectamine 2000(Invitrogen Inc.) according to the manufacturer's protocol. In order tohave large enough amounts of cells, one well of 6-well plate was at80-90% confluence for HEK293T, Gα16/HEK293T, Gqi5/HEK293T, CHO-K1,Gα16/CHO-K1 and Gqi5/CHO-K1 cells or one T25 at 90% confluence forRH7777, 1321N1 cells.

Flow cytometry analysis. To determine cell-surface GPCR expression,cells were detached from the plates with Cellstripper (Mediatech, Inc.).Cells were washed once with PBS, once with PBS/1% BSA at 4° C. They wereincubated with mouse anti-Flag M2 mAb (Sigma Chemical Co) for 30 minutesat 4° C., washed twice with PBS/1% BSA at 4° C., and further incubatedon ice in the dark with FITC-labeled goat anti-mouse IgG Sigma ChemicalCo) as secondary antibody. Cells were washed twice again and resuspendedin 400 μl PBS/1% BSA. The fluorescence of 10,000 cells/tube was assayedby a FACSort flow cytofluorometer (Becton Dickinson). For directimmunofluorescent staining, cells were first incubated with PE-labeledmouse anti-FLAG antibody (Prozyme Inc. San Leandro, Calif.) at 30 ug/mlfor 30 minutes on ice, then washed and analyzed as described above.

Stable Pool Selection and Isolation of Stable Cell Lines ExpressingHigh-Level GPCRs

To isolate stable populations, the cells were transfected in 6-wellplates, transferred to 100-mm Petri dish two days post transfection. Thestable pools for were selected by incubation of the cells in culturemedium containing 1 ug/ml puromycin (InvivoGen Inc., San Diego, Calif.)for another 10 to 14 days for HEK293 transfectants, or medium containing10 ug/ml puromycin for 7 to 10 days for CHOK1 transfectants.

To isolate single cells expressing high levels of the receptors, thestable colonies were pooled together, subjected to cell sorting withFACSort (Becton Dickinson) to separate the high expressers from the restof the population. The top 0.5% to top 5% high expressers in the totalstable population were gated and sorted out subsequently into sterile50-ml conical tubes pre-soaked with 4% BSA in PBS. The cells wereconcentrated by centrifugation and either plated on 96-well plates byserial dilution at 0.5, 1, and 5 cells/well, or plated in 150-mm Petridish in medium containing puromycin. Single-cell colonies were confirmedby examination under a microscope, expanded into 24-well plates, andanalyzed by FACS analysis. One such recombinant CHO clone, 9-4, whichexpresses high level of the human lysophosphatidic acid receptor 4(EDG4) in CHO-K1 cells is shown by FACS analysis in FIG. 18. The FACSprofile for the recombinant HEK293 cell for NMUR1 (colony 3) is shown inFIG. 17. The expression level of EDG4 in CHOK1 (clone 9-4) and NUMR1 inHEK293 (colony 3) are above 200,000 copies per cell, as measured usingcalibrated standardized phycoerythrin (PE)-conjugated beads (BDQuantiBRITE) and PE-conjugated M2 antibody.

The high-expressing cell lines exhibited good cellular response (such asintracellular calcium release) upon ligand binding to the receptors. Thedeveloped GPCR expressing cell lines provide critical tools forcell-based functional drug screening, in vitro ligand-binding assays,crystallization for structural studies and the development of monoclonalantibodies with the use of the recombinant whole cells as immunogens.Data for two representative cell lines are shown in FIG. 17.

Example V Antibody Development: Whole Cell Immunization Strategy

Immunization of animals GPCR expressing isogenic cell line at anexpression level of >100,000 copies per cell was used as immunogen.Female Balb/c mice of 6-8 weeks old are primary choice of animal. Eachmouse is immunized with 5-10 millions of live cells per mouse. Mouse:6-8 weeks Balb/c mouse as primary strain. Another strain with differentMHC II Type as backup: Live cells (about 10⁶ per mouse) plus Freund'sadjuvant. Cells are destroyed during emulsifying antigen.

On the first day, pre-immune bleeds are taken. Cells (about 20 million)are harvested and washed 3× with large volume (40 ml) of PBS, andre-suspended in PBS. Cell suspension is mixed with equal amount ofComplete Freund's adjuvant (CFA), emulsified and injected about 5millions of cells per mouse intraperitoneally.

The Day 1 procedure was repeated over the next few weeks (except noadjuvant every other 2 weeks) until a specific antibody titerof >1:50,000 is reached.

Tail bleeds were taken 7-10 days after the third, sixth and ninthimmunizations and will be tested by whole cell ELISA againstimmunogen-expressing and non-expressing cells. The cells used in wholecell ELISA ideally should be different from immunogen. Best responders(antibody titer>1:50,000) against desired antigen is expected.

Final boost was given to the best responder(s), 2.5 millions of cells ivand 2.5 millions of cells ip 3-4 days before the hybridoma fusion.

Titering of sera. Serum titers for individual mice within eachimmunization group were determined in duplicate against thecorresponding screening antigens (both cells expressing andnon-expressing desired GPCR). Groups of mice that failed to show titersafter extended periods of immunization will be terminated.

Lymphoid organ harvest. Immunoresponsive mice as determined by titeringwere used for hybridoma generation and the designated lymphoid organs(spleen in one embodiment) harvested and processed using conventionalpractices for B cell isolation.

Fusion. The fusion was done the same day following a standard hybridomafusion protocol. SP2/0 mouse myeloma cell line will be used as fusionpartner. The plated fusion products (10-20 plates per fusion) wereplated at intermediate density (not more than 10^7 cells per plate), andthen cultured for 11-14 days prior to screening.

Primary screening. Cell culture supernatants were tested against therelevant screening antigens with a whole cell ELISA protocol. Anti-mouseIgG antibody-enzyme reporter conjugate were used to detect antigenspecific immunoreactivity in the wells and identify those of interestfor retesting and potential cloning. Following data analysis, all strongpositive lineages were picked into 24-well cell culture plates.

Cell expansion. The cells in 24-well cell culture plates were culturedto exhaustion and cell culture supernatants (˜2 ml) were recovered toverify the original screening antigen reactivities.

Secondary Screening. All 24-well cell culture supernatants werere-tested against the relevant screening antigens following the protocolin Appendix 3 and with the desired applications, such as, Western Blot,ImmunoPrecipitation, Flow Cytometry and immunohistochemistry. Cell lineswere also banked at this stage. This activity at times required up to 14days. Following data analysis, line supernatants whose screening antigenreactivity were verified were advanced to subcloning.

Subcloning. Subcloning by limiting dilution for all hybridoma linesselected were conducted. If there were multiple clones that showedexactly the same functionalities, only the two best were cloned. Up to 3daughter clones were selected from each linage, based on visualinspection, production and immunoreactivity. Clones were screened withthe relevant screening antigen approximately 10-12 days post subcloning,depending on individual growth rates in culture. The completesubclonings and screening procedures required up to 4 weeks, dependinghow many rounds of subclonings were needed.

The final clones from previous work were processed to scaling up in thisphase through ascites generation. Antibody purification was neededdepending on the result of application.

Example VI Hybridoma Screening

ELISA plates (Corning 3369 or similar) were coated with 100 μl ofhigh-density (concentration to be decided) cells expressing desiredproteins (in PBS). The plates were allowed to air-dry inside a cellculture hood at room temperature for overnight. Negative control cells(non-transfected cells) were processed in parallel with the transfectedcells.

After overnight culture, the plates were washed three times withPBS+0.05% Tween-20 (PBST) and then blocked with 2501/well of PBST-5%skim milk). The plates were then incubated at room temperature for 1hour (or at 4° C. overnight).

After incubation, the PBST-5% skim Milk was discarded and 50 μl/wellcell culture supernatant or other form of testing antibodies was added,followed by another incubation for one hour at room temperature (orovernight at 4° C.).

After another 3× wash with PBST, 1:10,000 diluted goat anti-mouseIgG-HRP conjugate (Jackson Immuno 115-036-071 or similar) was added theplates incubated at room temperature for another hour.

After the incubation and a five time wash in PBST, HRP (horseradishperoxidase) substrate, Sigma Fast OPD was added and the plates incubatedin the dark at room temperature for 30-60 min.

Plates were read at OD450 with a 96-well colormetric detector ifreaction was not stopped, or at OD492 if stopped with 1.25M sulfuricacid.

Example VII FACS, Sorting Protocol for Establishing Stable Cell Line

This protocol was designed for use in conjunction with an anti-flag-tagantibody (clone M2 from Sigma-Aldrich) and the mammalian cellstransfected with pMEX plasmids carrying a full-length GPCR gene. Afraction of cells, 100K cells, were used to determine GPCR expressionlevel by FACS on FACSort (Becton Dickinson) following a96-well-microtiter-plate (use any U-shape plate) protocol below:

-   -   1. Resuspend cells and transfer them to a 15-ml centrifuge tube,        1200 rpm 5 min;    -   2. Wash cells once with 10 ml of cold PBS;    -   3. Wash cells with 10 ml of cold PBS+1% BSA (FB);    -   4. Resuspend the cells with cold FB and add 100 μl per well;    -   5. 1200 rpm 2 min, and flick the plate;    -   6. Vortex briefly to suspend the cells;    -   7. Add 100 μl of M2 antibody (anti-flag-tag) at 10 μg/ml;    -   8. Put the plate on top of ice for 30 min;    -   9. Spin-flick to remove M2 antibody;    -   10. Wash 2× with 250 μl FB;    -   11. Vortex, add to each well 100 μl of FITC-labeled antibodies        (anti-mouse) at about 10 μl per ml (1:100 dilution from a 1        mg/ml product);    -   12. Put the plate on top of ice for 20 min;    -   13. Spin-flick, then Wash 2× with 250 μl FB;    -   14. Resuspend cells in 250 μl FB and transfer cells to FACS        tubes;    -   15. Ready for FACS.

Two to three cells were collected. The collected cells were centrifugedand resuspended in selection medium, and immediately aliquoted 100 μl orone cell per well to 96-well plate. Cell number was estimated based onthe number of cells collected during the sorting.

Example VIII Protocol for Calcium Mobilization Assay

The Gq-coupled GPCRs expressed in CHO or HEK293T or other cells and theGPCRs coupled with other G-proteins were expressed in the cellstransfected with chimeric G-proteins. Functional expression was testedusing FLIPR Calcium 4 Assay Kit (Molecular Devices) using the standardprotocol, which is reproduced as follows:

A. Preparation of Cells

-   -   1. Culture adherent cells in 96-well ploy-D-lysine-coated        microplates (Sigma, cat# M-5307), to near confluence. CHO cells        can be plated at 30,000-40,000 cells per well and grown        overnight. HEK293 cells can be plated at 40,000-50,000 cells per        well and grown overnight.

B. Preparation of Reagents

-   -   2. Make a 250 mM stock of probenecid acid (100×): dissolve in 1        N NaOH and neutralize with equal volume of HBSS/HEPES.    -   3. Prepare the dye loading solution (for one microplate): add 10        ml of assay buffer and 100 μl of probenecid acid (final        concentration: 2.5 mM) stock solution to a vial of dye mix.        Vortex for 1 minute to ensure a complete dissolving.    -   4. Prepare a solution of receptor agonist (3×) in assay buffer        with 0.1% BSA. Make serial dilution in 96-well compound plate        (VWR #62409-112, NUNC, V-bottom)

C. Assay

-   -   5. Remove the growth medium from the adherent cell cultures.        Quickly but carefully add 100 μl of the dye loading solution to        each well of a 96-well plate.    -   6. Incubate the plate at 37° C. for 1 hour.    -   7. Measure fluorescence using Flexstation (Molecular Device).        Instrument settings: excitation at 485 nM, emission at 525 nM,        cut-off at 515 nM, compound addition (transfer volume): 50 μl,        addition speed (rate): 2, pipette height: 80 μl, assay duration        2-3 minutes.    -   HBSS/HEPES: Hanks' Balanced Salt Solution (1×) with 20 mM HEPES,        pH 7.4.

Although the invention has been described with reference to thepresently preferred embodiments, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

1. An expression vector comprising nucleotides 1-1197 of SEQ ID NO 19; anucleic acid sequence encoding a GPCR polypeptide; and nucleotides1643-5796 of SEQ ID NO: 19; wherein the expression vector facilitatesexpression of at least 150,000 copies of the GPCR polypeptide on thecytoplasmic membrane of a cell.
 2. The vector of claim 1, wherein theGPCR polypeptide is a member of a GPCR family selected from: ananaphylatoxin receptor, an apelin receptor, a bombesin receptor, acannabinoid receptor, a chemokine receptor, a free fatty acid receptor,a galanin receptor, a glucagon receptor, a glycoprotein hormonereceptor, a leukotriene/lipoxin receptor, a lysophospholipid receptor, amelanin-concentrating hormone receptor, a melatonin receptor, aN-formylpeptide receptor, a neuromedin U receptor, a neuropeptide Sreceptor, a neuropeptide W/neuropeptide B receptor, a neuropeptide Yreceptor, an opioid receptor, a platelet activating factor receptor, aprolactin releasing peptide receptor, a prostanoid receptor, a PTHreceptor, a purinergic receptor, a tachykinin receptor, a trace aminereceptor, and a urotensin receptor.
 3. The vector of claim 1, whereinthe GPCR polypeptide is an orphan GPCR.
 4. The vector of claim 1,wherein the GPCR polypeptide is selected from: C3aR, APJ, BB1, BB3,GPR55, CCR1, CCR5, CCR7, CCR9, CMKLR1, CXCR3, CXCR4, FFA1, FFA2, GAL1,GAL2, GAL3, GHRH, TSH, ALX, BLT1, BLT2, CysLT1, LPA2, LPA3, MCH1, MT2,FPR1, NMU1, NPS, NPS(1), NPS(2), NPS Ile107, NPBW1, NPBW2, delta, kappa,mu, NOP, GPR37L1, GPR84, MRGX1, MRGX2, PSGR, PAF, PRP, DP, EP1, GPR44,PTH2, P2Y12, NK2, NK3, TM, C5AR, and PAR2.
 5. The vector of claim 1,wherein the GPCR polypeptide comprises an amino acid sequence selectedfrom SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, and SEQ ID NO:
 17. 6.The vector of claim 1, wherein said nucleic acid sequence encoding theGPCR polypeptide is selected from: SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 6, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, andSEQ ID NO:
 18. 7. An expression vector comprising SEQ ID NO:19.
 8. Anisolated recombinant cell comprising: an expression vector comprisingnucleotides 1-1197 of SEQ ID NO 19; a nucleic acid sequence encoding aGPCR polypeptide; and nucleotides 1643-5796 of SEQ ID NO: 19; whereinthe expression vector facilitates expression of at least 150,000 copiesof the GPCR polypeptide on the cytoplasmic membrane of the cell.
 9. Therecombinant cell of claim 8, wherein said cell expresses said GPCRpolypeptide at a range of 150,000 copies to 2,000,000 copies per cell.10. The recombinant cell of claim 8, wherein said cell expresses saidGPCR polypeptide at a range of 200,000 copies to 2,000,000 copies percell.
 11. The recombinant cell of claim 8, wherein said cell expressessaid GPCR polypeptide at a range of 400,000 copies to 2,000,000 copiesper cell.
 12. The recombinant cell of claim 8, wherein said cellexpresses said GPCR polypeptide at a range of 800,000 copies to2,000,000 copies per cell.
 13. The recombinant cell of claim 8, whereinsaid cell expresses said GPCR polypeptide at a range of 1,000,000 copiesto 2,000,000 copies per cell.
 14. The recombinant cell of claim 8,wherein said cell expresses said GPCR polypeptide at a range of1,500,000 copies to 2,000,000 copies per cell.
 15. The recombinant cellof claim 8, wherein said cell is a member selected from the group CHO,HEK293T, C6, RH7777, SW480, VS35, and 1321N1 cells.
 16. The vector ofclaim 8, wherein the GPCR polypeptide comprises an amino acid sequenceselected from SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 5, SEQ ID NO: 7, SEQID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, and SEQ ID NO:17.
 17. The recombinant cell of claim 8, wherein said nucleic acidsequence encoding the GPCR polypeptide is selected from: SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 14,SEQ ID NO: 16, and SEQ ID NO:
 18. 18. A method of producing a GPCRpolypeptide, said method comprising culturing the recombinant cell ofclaim 8 in vitro under conditions suitable for expression of the GPCRpolypeptide; and recovering the GPCR polypeptide.
 19. The method ofclaim 18, wherein said cell is a mammalian cell.
 20. The method of claim18, wherein said cell is selected from CHO, HEK293T, C6, RH7777, SW480,VS35, and 1321N1.
 21. The method of claim 18, wherein said nucleic acidsequence encoding the GPCR polypeptide is selected from: SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 14,SEQ ID NO: 16, and SEQ ID NO:
 18. 22. A method of screening for atherapeutic candidate comprising: culturing the recombinant cell ofclaim 10 in vitro under conditions suitable for expression of the GPCRpolypeptide; b) contacting a test entity with a region of said GPCRpolypeptide, wherein said region presents a fragment of said GPCRpolypeptide sufficient for said test entity to interact detectably withsaid fragment; and c) detecting interaction of said test entity withsaid fragment, thereby identifying said test entity as said therapeuticcandidate.
 23. The method of claim 22, wherein detecting interaction ofsaid test entity with said fragment comprises use of a fluorescent,chemical, radiological, or enzymatic reporter.
 24. The method of claim22, wherein said test entity is contacted with a membrane extract ofsaid recombinant cell.
 25. The method of claim 22, wherein said methodemploys a high, throughput screen for detecting interaction of said testentity with said fragment.
 26. A method for producing a functional assaycell line comprising: a) producing a cell line comprising therecombinant cell of claim 8 by culturing the cell in vitro underconditions suitable for expression of the GPCR polypeptide; b) couplinga functional reporter to the binding of a ligand to said GPCRpolypeptide, such that binding between said ligand and said GPCRpolypeptide is detectable as a reporter activity readout.
 27. The methodof claim 26, wherein said reporter activity readout comprises detectionof second messenger activity.
 28. The method of claim 27, wherein saidsecond messenger activity comprises: a change in intracellular calciumlevels, cAMP activity, and NFAT or CRE, driven beta-lactamase.
 29. Themethod of claim 26, wherein said reporter activity readout comprisesdetection of GFP, luciferase, or a radio-labeled molecule.
 30. A methodof using a GPCR-expressing cell line to identify a test compound whichmodulates activity of said GPCR comprising: a) producing a cell linecomprising the recombinant cell of claim 8 by culturing the cell invitro under conditions suitable for expression of the GPCR polypeptide;b) measuring second messenger activity in said cell line in the absenceof said test compound, thereby obtaining a first measurement; c)measuring second messenger activity in said cell line in the presence ofsaid test compound, thereby obtaining a second measurement; and d)comparing said first measurement and said second measurement andidentifying those compounds that result in a difference between saidfirst measurement and said second measurement as test compounds thatmodulate the activity of said GPCR.
 31. The method of claim 30, whereinsaid second messenger activity comprises a rise in intracellularcalcium.
 32. The method of claim 30, wherein said second messengeractivity comprises a change in intracellular cAMP levels.
 33. The methodof claim 30, wherein said method includes high, throughput screeningmethods to detect said second messenger activity.